Publications by authors named "Shaheen Khan"

144 Publications

An initial analysis of the UK Medical Cannabis Registry: Outcomes analysis of first 129 patients.

Neuropsychopharmacol Rep 2021 May 14. Epub 2021 May 14.

Imperial College London, London, UK.

Aim: Cannabis-based medicinal products (CBMPs) are prescribed with increased frequency, despite a paucity of high-quality randomized controlled trials. The aim of this study is to analyze the early outcomes of the first series of patients prescribed CBMPs in the UK with respect to effects on health-related quality of life and clinical safety.

Methods: A prospective case series was performed using the UK Medical Cannabis Registry. Primary outcomes were change in patient-reported outcomes measures (EQ-5D-5L, General Anxiety Disorder-7 (GAD-7) and Single-Item Sleep Quality Scale (SQS)) at 1 and 3 months from baseline. The secondary outcome was the incidence of adverse events. Statistical significance was defined by a P-value <.050.

Results: There were 129 patients included in the final analysis with a mean age of 46.23 (±14.51) years. The most common indication was chronic pain of undefined etiology (n = 48; 37.2%). The median initial cannabidiol and (-)-trans-Δ⁹-tetrahydrocannabinol daily dose was 20.0 mg (Range: 0.0-768.0 mg) and 3.9 mg (Range: 0.0-660.0 mg), respectively. Statistically significant improvements in health-related quality of life were demonstrated at 1 and 3 months in GAD-7, SQS, EQ-5D-5L pain and discomfort subscale, EQ-5D-5L anxiety and depression subscale, EQ-VAS and EQ-5D-5L index values(P < .050). There were 31 (24.03%) total reported adverse events.

Conclusion: This study suggests that CBMP therapy may be associated with an improvement in health-related quality-of-life outcomes as self-reported by patients. CBMPs are also demonstrated to be relatively safe in the short to medium-term. These findings must be treated with caution given the limited scope of this initial analysis, with no placebo or an active comparator, with further research required.
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http://dx.doi.org/10.1002/npr2.12183DOI Listing
May 2021

Melanoma models for the next generation of therapies.

Cancer Cell 2021 May 4;39(5):610-631. Epub 2021 Feb 4.

Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health, and Department of Oncology, Sidney Kimmel Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA.

There is a lack of appropriate melanoma models that can be used to evaluate the efficacy of novel therapeutic modalities. Here, we discuss the current state of the art of melanoma models including genetically engineered mouse, patient-derived xenograft, zebrafish, and ex vivo and in vitro models. We also identify five major challenges that can be addressed using such models, including metastasis and tumor dormancy, drug resistance, the melanoma immune response, and the impact of aging and environmental exposures on melanoma progression and drug resistance. Additionally, we discuss the opportunity for building models for rare subtypes of melanomas, which represent an unmet critical need. Finally, we identify key recommendations for melanoma models that may improve accuracy of preclinical testing and predict efficacy in clinical trials, to help usher in the next generation of melanoma therapies.
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http://dx.doi.org/10.1016/j.ccell.2021.01.011DOI Listing
May 2021

HCV Replicon Systems: Workhorses of Drug Discovery and Resistance.

Front Cell Infect Microbiol 2020 30;10:325. Epub 2020 Jun 30.

Virology Section, Department of Life Sciences, Shiv Nadar University, Gautam Buddha Nagar, India.

The development of direct-acting antivirals (DAAs) has revolutionized the state-of-the art treatment of HCV infections, with sustained virologic response rates above 90%. However, viral variants harboring substitutions referred to as resistance-associated substitutions (RASs) may be present in baseline levels and confer resistance to DAAs, thereby posing a major challenge for HCV treatment. HCV replicons have been the primary tools for discovering and evaluating the inhibitory activity of DAAs against viral replication. Interest in replicon systems has further grown as they have become indispensable for discovering genotype-specific and cross-genotype RASs. Here, we review functional replicon systems for HCV, how these replicon systems have contributed to the development of DAAs, and the characteristics and distribution of RASs for DAAs.
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http://dx.doi.org/10.3389/fcimb.2020.00325DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7344236PMC
June 2020

Statin Intolerance, Anti-HMGCR Antibodies, and Immune Checkpoint Inhibitor-Associated Myositis: A "Two-Hit" Autoimmune Toxicity or Clinical Predisposition?

Oncologist 2020 08 3;25(8):e1242-e1245. Epub 2020 Jun 3.

Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

Immune-related adverse events induced by immune checkpoint inhibitor (ICI) therapy may affect diverse organ systems, including skeletal and cardiac muscle. ICI-associated myositis may result in substantial morbidity and occasional mortality. We present a case of a patient with advanced non-small cell lung cancer who developed grade 4 myositis with concurrent myocarditis early after initiation of anti-programmed death ligand 1 therapy (durvalumab). Autoantibody analysis revealed marked increases in anti-3-hydroxy-3-methylglutaryl-coenzyme A reductase antibody levels that preceded clinical toxicity, and further increased during toxicity. Notably, the patient had a history of intolerable statin myopathy, which had resolved clinically after statin discontinuation and prior to ICI initiation. This case demonstrates a potential association between statin exposure, autoantibodies, and ICI-associated myositis.
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http://dx.doi.org/10.1634/theoncologist.2019-0911DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7418340PMC
August 2020

Investigational Biomarkers for Checkpoint Inhibitor Immune-Related Adverse Event Prediction and Diagnosis.

Clin Chem 2020 06;66(6):779-793

Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX.

Background: Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of multiple cancers. However, these promising therapies may also cause immune-related adverse events (irAEs) in a substantial proportion of patients. These autoimmune phenomena may affect almost any organ system and may occur at almost any point in therapy. In some instances, these toxicities are life-threatening and potentially permanent. Diverse clinical presentation and unpredictable timing further complicate their anticipation and diagnosis.

Content: To improve patient safety and selection for ICI use, biomarkers for irAE diagnosis and prediction are under development. Clinicians may use traditional laboratory markers such as routine chemistries, creatinine clearance, thyroid function tests, and serum cortisol/adrenocorticotrophic hormone to monitor for specific irAEs, but noted aberrations may not necessarily represent an immune-mediated etiology. Novel biomarkers have the potential to be more specific to assist in the diagnosis of irAEs. The prediction of irAEs is more challenging. Apart from a history of autoimmune disease, no other clinical parameters are routinely used to project risk. Biomarker candidates under investigation for irAE diagnosis and prediction include blood cell analysis, chemokines/cytokines, autoantibodies, and genetic predisposition, such as human leukocyte antigen haplotype. Among other emerging candidates are immune-cell subsets, T-cell repertoire, fecal microbiome, tumor genomics, and radiomic characterization.

Summary: Several conventional laboratory indexes of end-organ dysfunction are currently in routine clinical use for irAE monitoring and diagnosis. Novel biomarkers for the prediction and diagnosis of these irAEs, which primarily characterize patient immune function, represent an area of active investigation.
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http://dx.doi.org/10.1093/clinchem/hvaa081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7259479PMC
June 2020

Late-Onset Immunotherapy Toxicity and Delayed Autoantibody Changes: Checkpoint Inhibitor-Induced Raynaud's-Like Phenomenon.

Oncologist 2020 05 13;25(5):e753-e757. Epub 2020 Mar 13.

Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

Immune checkpoint inhibitor (ICI)-induced immune-related adverse events (irAEs) may affect almost any organ system and occur at any point during therapy. Autoantibody analysis may provide insight into the mechanism, nature, and timing of these events. We report a case of ICI-induced late-onset Raynaud's-like phenomenon in a patient receiving combination immunotherapy. A 53-year-old woman with advanced non-small lung cancer received combination anti-cytotoxic T-lymphocyte antigen 4 and anti-programmed death 1 ICI therapy. She developed early (hypophysitis at 4 months) and late (Raynaud's at >20 months) irAEs. Longitudinal assessment of 124 autoantibodies was correlated with toxicity. Although autoantibody levels were generally stable for the first 18 months of therapy, shortly before the development of Raynaud's, a marked increase in multiple autoantibodies was observed. This case highlights the potential for delayed autoimmune toxicities and provides potential biologic insights into the dynamic nature of these events. KEY POINTS: A patient treated with dual anti-PD1 and anti-CTLA4 therapy developed Raynaud's-like signs and symptoms more than 18 months after starting therapy. In this case, autoantibody changes became apparent shortly before onset of clinical toxicity. This case highlights the potential for late-onset immune-related adverse events checkpoint inhibitors, requiring continuous clinical vigilance. The optimal duration of checkpoint inhibitor therapy in patients with profound and prolonged responses remains unclear.
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http://dx.doi.org/10.1634/theoncologist.2019-0666DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7216445PMC
May 2020

Genome-Wide Mutagenesis of Hepatitis C Virus Reveals Ability of Genome To Overcome Detrimental Mutations.

J Virol 2020 01 17;94(3). Epub 2020 Jan 17.

Virology Section, Department of Life Sciences, Shiv Nadar University, Gautam Buddha Nagar, Uttar Pradesh, India

To gain insight into the impact of mutations on the viability of the hepatitis C virus (HCV) genome, we created a set of full-genome mutant libraries, differing from the parent sequence as well as each other, by using a random mutagenesis approach; the proportion of mutations increased across these libraries with declining template amount or dATP concentration. The replication efficiencies of full-genome mutant libraries ranged between 71 and 329 focus-forming units (FFU) per 10 Huh7.5 cells. Mutant libraries with low proportions of mutations demonstrated low replication capabilities, whereas those with high proportions of mutations had their replication capabilities restored. Hepatoma cells transfected with selected mutant libraries, with low (4 mutations per 10,000 bp copied), moderate (33 mutations), and high (66 mutations) proportions of mutations, and their progeny were subjected to serial passage. Predominant virus variants (mutants) from these mutant libraries (Mutant, Mutant, and Mutant, respectively) were evaluated for changes in growth kinetics and particle-to-FFU unit ratio, virus protein expression, and modulation of host cell protein synthesis. Mutant and Mutant variants produced >3.0-log-higher extracellular progeny per ml than the parent, and Mutant produced progeny at a rate 1.0-log lower. More than 80% of the mutations were in a nonstructural part of the mutant genomes, the majority were nonsynonymous, and a moderate to large proportion were in the conserved regions. Our results suggest that the HCV genome has the ability to overcome lethal/deleterious mutations because of the high reproduction rate but highly selects for random, beneficial mutations. Hepatitis C virus (HCV) displays high genetic heterogeneity, which is partly due to the high reproduction and random substitutions during error-prone genome replication. It is difficult to introduce random substitutions because of limitations in inducing mutagenesis from the 5' end to the 3' end of the genome. Our study has overcome this limitation. We synthesized full-length genomes with few to several random mutations in the background of an HCV clone that can recapitulate all steps of the life cycle. Our study provides evidence of the capability of the HCV genome to overcome deleterious mutations and remain viable. Mutants that emerged from the libraries had diverse phenotype profiles compared to the parent, and putative adaptive mutations mapped to segments of the conserved nonstructural genome. We demonstrate the potential utility of our system for the study of sequence variation that ensures the survival and adaptation of HCV.
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http://dx.doi.org/10.1128/JVI.01327-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7000968PMC
January 2020

FOXN1 compound heterozygous mutations cause selective thymic hypoplasia in humans.

J Clin Invest 2019 11;129(11):4724-4738

Departments of Immunology, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

We report on 2 patients with compound heterozygous mutations in forkhead box N1 (FOXN1), a transcription factor essential for thymic epithelial cell (TEC) differentiation. TECs are critical for T cell development. Both patients had a presentation consistent with T-/loB+NK+ SCID, with normal hair and nails, distinct from the classic nude/SCID phenotype in individuals with autosomal-recessive FOXN1 mutations. To understand the basis of this phenotype and the effects of the mutations on FOXN1, we generated mice using CRISPR-Cas9 technology to genocopy mutations in 1 of the patients. The mice with the Foxn1 compound heterozygous mutations had thymic hypoplasia, causing a T-B+NK+ SCID phenotype, whereas the hair and nails of these mice were normal. Characterization of the functional changes due to the Foxn1 mutations revealed a 5-amino acid segment at the end of the DNA-binding domain essential for the development of TECs but not keratinocytes. The transcriptional activity of this Foxn1 mutant was partly retained, indicating a region that specifies TEC functions. Analysis of an additional 9 FOXN1 mutations identified in multiple unrelated patients revealed distinct functional consequences contingent on the impact of the mutation on the DNA-binding and transactivation domains of FOXN1.
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http://dx.doi.org/10.1172/JCI127565DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6819092PMC
November 2019

Autoimmunity, checkpoint inhibitor therapy and immune-related adverse events: A review.

Semin Cancer Biol 2020 08 19;64:93-101. Epub 2019 Jul 19.

Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, 75390-9093, United States; Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX, 75390-9093, United States; Department of Population & Data Sciences, University of Texas Southwestern Medical Center, Dallas, TX, 75390-9093, United States. Electronic address:

Immune checkpoint inhibitors have emerged as a remarkable treatment option for diverse cancer types. However, a significant number of patients on checkpoint inhibitors develop immune-related adverse events (irAEs) affecting a wide variety of organs. These events, which may reflect enhanced T cell activation, are unpredictable, heterogeneous, and in some instances permanent or life-threatening. It is not clear whether these toxicities are distinct from conventional autoimmune diseases or whether the manifestation of irAEs is associated with therapeutic efficacy. Studies across the spectrum of basic, preclinical and clinical research deciphering the role of genetics, epigenetics, gut microbiota and underlying immune status of patients who develop irAEs are required to gain a deeper mechanistic understanding. Insights gained from such studies will facilitate identification of biomarkers for optimal treatment and clinical management of patients. In this Review, we provide basic and clinical understanding of immune checkpoint inhibitors and irAEs. We discuss the connection between immune system, autoimmunity and cancer; immune checkpoint inhibitors and associated autoimmune toxicities; insights into potential underlying mechanisms of irAEs; impact of autoimmune diagnosis on cancer outcome; and management of irAEs.
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http://dx.doi.org/10.1016/j.semcancer.2019.06.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6980444PMC
August 2020

Single-Cell RNA-Sequencing of Peripheral Blood Mononuclear Cells with ddSEQ.

Methods Mol Biol 2019 ;1979:155-176

Digital Biology Center, Bio-Rad Laboratories, Pleasanton, CA, USA.

Peripheral blood mononuclear cells (PBMCs) are blood cells that are a critical part of the immune system used to fight off infection. However, due to the complexity of PBMCs, which contain multiple different cell types, studying the function of the individual cell types can be difficult, and often studies rely on bulk measurements. Here, we describe the analysis of PBMCs using single-cell RNA-sequencing in droplets. Data from these studies allow for the identification and quantification of the subpopulation of cells that make up the PBMC sample. In addition, differential gene expression between cell types and samples can be assessed.
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http://dx.doi.org/10.1007/978-1-4939-9240-9_10DOI Listing
August 2019

MIR205HG Is a Long Noncoding RNA that Regulates Growth Hormone and Prolactin Production in the Anterior Pituitary.

Dev Cell 2019 05 11;49(4):618-631.e5. Epub 2019 Apr 11.

Department of Immunology University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA; Department of Microbiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA; Department of Pediatrics, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA. Electronic address:

MicroRNAs (miRNAs) are processed from primary miRNA transcripts (pri-miRNAs), many of which are annotated as long noncoding RNAs (lncRNAs). We assessed whether MIR205HG, the host gene for miR-205, has independent functions as an lncRNA. Comparing mice with targeted deletions of MIR205HG and miR-205 revealed a functional role for the lncRNA in the anterior pituitary. Mice lacking MIR205HG had a temporal reduction in Pit1, growth hormone, and prolactin. This was mediated, in part, through the ability of this lncRNA to bind and regulate the transcriptional activity of Pit1 in conjunction with Zbtb20. Knockdown of MIR205HG in lactotropes decreased the expression of Pit1, Zbtb20, prolactin, and growth hormone, while its overexpression enhanced the levels of these transcripts. The effects of MIR205HG on the pituitary were independent of miR-205. The data support a role for MIR205HG as an lncRNA that regulates growth hormone and prolactin production in the anterior pituitary.
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http://dx.doi.org/10.1016/j.devcel.2019.03.012DOI Listing
May 2019

Human neonatal stem cell-derived skin substitute improves healing of severe burn wounds in a rat model.

Cell Biol Int 2019 Feb;43(2):147-157

National Center of Excellence in Molecular Biology (CEMB), University of the Punjab, Lahore, Pakistan.

Conventional approaches can repair minor skin injuries; however, severe burn injuries require innovative approaches for efficient and better wound repair. Recent studies indicate that stem cell-based regenerative therapies can restore severe damaged skin both structurally and functionally. The current study aims to evaluate the wound healing potential of skin substitute derived from human neonatal stem cells (hNSCs) using a severe burn injury rat model. Amniotic epithelial cells (AECs) and mesenchymal stem cells (MSCs) were isolated from placenta (a source of neonatal stem cells) by explant culture method. After characterization, AECs and umbilical cord-MSCs were differentiated into keratinocyte and fibroblasts, respectively. Morphological changes, and expression of corresponding keratinocyte and fibroblast specific markers were used to verify differentiation into respective lineage. A skin substitute was developed by mixing hNSCs-derived skin cells (hNSCs-SCs) in plasma for transplantation in a rat model of severe burn injury. Results indicated that placenta-derived AECs and MSCs were efficiently differentiated into skin cells, that is, keratinocytes and fibroblasts, respectively, as indicated by morphological changes, immunostaining, and polymerase chain reaction analysis. Further, transplantation of hNSCs-SCs seeded in plasma significantly improved basic skin architecture, re-epithelization rate, and wound healing concurrent with reduced apoptosis. In conclusion, neonatal stem cell-derived skin substitute efficiently improved severe burn wounds in a rat model of burn injury. Unique properties of placenta-derived stem cells make them superlative candidates for the development of "off-the-shelf" artificial skin substitutes for future use.
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http://dx.doi.org/10.1002/cbin.11072DOI Listing
February 2019

Immune dysregulation in cancer patients developing immune-related adverse events.

Br J Cancer 2019 01 31;120(1):63-68. Epub 2018 Oct 31.

Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, 75390-9093, USA.

Background: Up to 40% of cancer patients on immune checkpoint inhibitors develop clinically significant immune-related adverse events (irAEs). The role of host immune status and function in predisposing patients to the development of irAEs remains unknown.

Methods: Sera from 65 patients receiving immune checkpoint inhibitors and 13 healthy controls were evaluated for 40 cytokines at pre-treatment, after 2-3 weeks and after 6 weeks and analysed for correlation with the development of irAEs.

Results: Of the 65 cancer patients enrolled, 55% were women; the mean age was 65 years and 98% received anti-PD1/PDL1 therapy. irAEs occurred in 35% of cases. Among healthy controls, cytokine levels were stable over time and lower than those in cancer patients at baseline. Significant increases in CXCL9, CXCL10, CXCL11 and CXCL13 occurred 2 weeks post treatment, and in CXCL9, CXCL10, CXCL11, CXCL13, IL-10 and CCL26 at 6 weeks post treatment. Patients who developed irAEs had lower levels of CXCL9, CXCL10, CXCL11 and CXCL19 at baseline and exhibited greater increases in CXCL9 and CXCL10 levels at post treatment compared to patients without irAEs.

Conclusions: Patients who developed irAEs have lower baseline levels and greater post-treatment increases in multiple cytokine levels, suggesting that underlying immune dysregulation may be associated with heightened risk for irAEs.
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http://dx.doi.org/10.1038/s41416-018-0155-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6325132PMC
January 2019

Global genetic insight contributed by consanguineous Pakistani families segregating hearing loss.

Hum Mutat 2019 01 18;40(1):53-72. Epub 2018 Nov 18.

Department of Otorhinolaryngology-Head and Neck Surgery, School of Medicine, University of Maryland, Baltimore, Maryland.

Consanguineous Pakistani pedigrees segregating deafness have contributed decisively to the discovery of 31 of the 68 genes associated with nonsyndromic autosomal recessive hearing loss (HL) worldwide. In this study, we utilized genome-wide genotyping, Sanger and exome sequencing to identify 163 DNA variants in 41 previously reported HL genes segregating in 321 Pakistani families. Of these, 70 (42.9%) variants identified in 29 genes are novel. As expected from genetic studies of disorders segregating in consanguineous families, the majority of affected individuals (94.4%) are homozygous for HL-associated variants, with the other variants being compound heterozygotes. The five most common HL genes in the Pakistani population are SLC26A4, MYO7A, GJB2, CIB2 and HGF, respectively. Our study provides a profile of the genetic etiology of HL in Pakistani families, which will allow for the development of more efficient genetic diagnostic tools, aid in accurate genetic counseling, and guide application of future gene-based therapies. These findings are also valuable in interpreting pathogenicity of variants that are potentially associated with HL in individuals of all ancestries. The Pakistani population, and its infrastructure for studying human genetics, will continue to be valuable to gene discovery for HL and other inherited disorders.
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http://dx.doi.org/10.1002/humu.23666DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6296877PMC
January 2019

Human amniotic membrane as differentiating matrix for in vitro chondrogenesis.

Regen Med 2018 10 9;13(7):821-832. Epub 2018 Oct 9.

Centre of Excellence in Molecular Biology, 87 West Canal Bank Road, Thokar Niazbaig Lahore, Punjab, 53700 Pakistan.

Aim: The aim of the present study is to use human amniotic membrane (HAM) for in vitro chondrogenesis of placenta-derived mesenchymal stem cells (MSCs) and umbilical cord-derived MSCs.

Materials & Methods: MSCs from the placenta and umbilical cord were isolated, characterized by immunophenotyping and after analyzing their rate of proliferation, cytotoxicity and viability, chondrogenesis was performed on plastic adherent surface and on HAM.

Results: Successfully isolated and characterized placenta-derived MSCs and umbilical cord-derived MSCs revealed positive expression of MSCs markers CD90, CD73, CD105 and CD49d, while they were negative for CD45. Both types of cells in the presence of chondrogenic induction medium on plastic adherent surface and HAM showed aggregates of proteoglycan and strong expression of COL2A1 (collagen 2) and ACAN1 (aggrecan).

Conclusion: HAM supported proliferation as well as chondrogenesis of MSCs and provide novelty of HAM utilization as an efficient natural delivery matrix for stem cell transplantation.
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http://dx.doi.org/10.2217/rme-2018-0017DOI Listing
October 2018

preconditioning of insulin-producing cells with growth factors improves their survival and ability to release insulin.

J Biosci 2018 Sep;43(4):649-659

National Centre of Excellence in Molecular Biology, University of Punjab, 87-West Canal Bank Road, Lahore, Pakistan.

Glucose-induced oxidative stress in the diabetic pancreas directly affects viability and the consequent therapeutic outcome of transplanted stem cells. Pretreatment of stem cells with growth factors induces tolerance in them against various stresses (hypoxia, thermal or hyperglycaemic). This study investigated the effect of pretreatment on insulin-producing cells (IPCs) differentiated from adipose-derived mesenchymal stem cells (ADMSCs), with a combination of stromal cell-derived factor 1 alpha (SDF1 α) and basic fibroblast growth factor (bFGF) against hyperglycaemic stress (17 or 33 mM glucose). The results showed that IPCs pretreated with a combination of SDF1α and bFGF exhibited maximally alleviated apoptosis, senescence and cell damage with a concomitantly increased release of insulin, enhanced cell proliferation and greater upregulation of Insulin 1, Insulin 2, Ngn3, Pdx1 and Nkx6.2 when stressed with 33 mM glucose. These findings may offer an improved therapeutic outcome for the treatment of diabetes.
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September 2018

Whole genome sequencing data for two individuals of Pakistani descent.

Sci Data 2018 09 11;5:180174. Epub 2018 Sep 11.

The Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA.

Here we report next-generation based whole genome sequencing of two individuals (H1 and H2) from a family of Pakistani descent. The genomic DNA was used to prepare paired-end libraries for whole-genome sequencing. Deep sequencing yielded 706.49 and 778.12 million mapped reads corresponding to 70.64 and 77.81 Gb sequence data and 23× and 25× average coverage for H1 and H2, respectively. Notably, a total of 448,544 and 470,683 novel variants, not present in the single nucleotide polymorphism database (dbSNP), were identified in H1 and H2, respectively. Comparative analysis identified 2,415,852 variants common in both genomes including 240,181 variants absent in the dbSNP. Principal component analysis linked the ancestry of both genomes with South Asian populations. In conclusion, we report whole genome sequences of two individuals from a family of Pakistani descent.
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http://dx.doi.org/10.1038/sdata.2018.174DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6137601PMC
September 2018

Publisher Correction: FBP17 and CIP4 recruit SHIP2 and lamellipodin to prime the plasma membrane for fast endophilin-mediated endocytosis.

Nat Cell Biol 2018 Oct;20(10):1229

Institute of Structural and Molecular Biology, University College London, London, UK.

In the version of this Letter originally published, the name of co-author Safa Lucken-Ardjomande Häsler was coded wrongly, resulting in it being incorrect when exported to citation databases. This has been corrected, though no visible changes will be apparent.
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http://dx.doi.org/10.1038/s41556-018-0191-3DOI Listing
October 2018

FBP17 and CIP4 recruit SHIP2 and lamellipodin to prime the plasma membrane for fast endophilin-mediated endocytosis.

Nat Cell Biol 2018 09 30;20(9):1023-1031. Epub 2018 Jul 30.

Institute of Structural and Molecular Biology, University College London, London, UK.

Endocytosis mediates the cellular uptake of micronutrients and the turnover of plasma membrane proteins. Clathrin-mediated endocytosis is the major uptake pathway in resting cells, but several clathrin-independent endocytic routes exist in parallel. One such pathway, fast endophilin-mediated endocytosis (FEME), is not constitutive but triggered upon activation of certain receptors, including the β adrenergic receptor. FEME activates promptly following stimulation as endophilin is pre-enriched by the phosphatidylinositol-3,4-bisphosphate-binding protein lamellipodin. However, in the absence of stimulation, endophilin foci abort and disassemble after a few seconds. Looking for additional proteins involved in FEME, we found that 20 out of 65 BAR domain-containing proteins tested colocalized with endophilin spots. Among them, FBP17 and CIP4 prime the membrane of resting cells for FEME by recruiting the 5'-lipid phosphatase SHIP2 and lamellipodin to mediate the local production of phosphatidylinositol-3,4-bisphosphate and endophilin pre-enrichment. Membrane-bound GTP-loaded Cdc42 recruits FBP17 and CIP4, before being locally deactivated by RICH1 and SH3BP1 GTPase-activating proteins. This generates the transient assembly and disassembly of endophilin spots, which lasts 5-10 seconds. This mechanism periodically primes patches of the membrane for prompt responses upon FEME activation.
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http://dx.doi.org/10.1038/s41556-018-0146-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6122583PMC
September 2018

Whole-Exome Sequencing Identifies Novel Variants that Co-segregates with Autosomal Recessive Retinal Degeneration in a Pakistani Pedigree.

Adv Exp Med Biol 2018 ;1074:219-228

Shiley Eye Institute, University of California San Diego, La Jolla, CA, USA.

Purpose: To identify the molecular basis of inherited retinal degeneration (IRD) in a familial case of Pakistani origin using whole-exome sequencing.

Methods: A thorough ophthalmic examination was completed, and genomic DNA was extracted using standard protocols. Whole exome(s) were captured with Agilent V5 + UTRs probes and sequenced on Illumina HiSeq genome analyzer. The exomeSuite software was used to filter variants, and the candidate causal variants were prioritized, examining their allele frequency and PolyPhen2, SIFT, and MutationTaster predictions. Sanger dideoxy sequencing was performed to confirm the segregation with disease phenotype and absence in ethnicity-matched control chromosomes.

Results: Ophthalmic examination confirmed retinal degeneration in all affected individuals that segregated as an autosomal recessive trait in the family. Whole-exome sequencing identified two homozygous missense variants: c.1304G > A; p.Arg435Gln in ZNF408 (NM_024741) and c.902G > A; p.Gly301Asp in C1QTNF4 (NM_031909). Both variants segregated with the retinal phenotype in the PKRD320 and were absent in ethnically matched control chromosomes.

Conclusion: Whole-exome sequencing coupled with bioinformatics analysis identified potential novel variants that might be responsible for IRD.
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http://dx.doi.org/10.1007/978-3-319-75402-4_27DOI Listing
May 2019

Mutations in Diphosphoinositol-Pentakisphosphate Kinase PPIP5K2 are associated with hearing loss in human and mouse.

PLoS Genet 2018 03 28;14(3):e1007297. Epub 2018 Mar 28.

Laboratory of Molecular Genetics, Department of Otorhinolaryngology-Head & Neck Surgery, School of Medicine University of Maryland, Baltimore, MD, United States of America.

Autosomal recessive nonsyndromic hearing loss is a genetically heterogeneous disorder. Here, we report a severe-to-profound sensorineural hearing loss locus, DFNB100 on chromosome 5q13.2-q23.2. Exome enrichment followed by massive parallel sequencing revealed a c.2510G>A transition variant in PPIP5K2 that segregated with DFNB100-associated hearing loss in two large apparently unrelated Pakistani families. PPIP5Ks enzymes interconvert 5-IP7 and IP8, two key members of the inositol pyrophosphate (PP-IP) cell-signaling family. Their actions at the interface of cell signaling and bioenergetic homeostasis can impact many biological processes. The c.2510G>A transition variant is predicted to substitute a highly invariant arginine residue with histidine (p.Arg837His) in the phosphatase domain of PPIP5K2. Biochemical studies revealed that the p.Arg837His variant reduces the phosphatase activity of PPIP5K2 and elevates its kinase activity. We found that in mouse inner ear, PPIP5K2 is expressed in the cochlear and vestibular sensory hair cells, supporting cells and spiral ganglion neurons. Mice homozygous for a targeted deletion of the Ppip5k2 phosphatase domain exhibit degeneration of cochlear outer hair cells and elevated hearing thresholds. Our demonstration that PPIP5K2 has a role in hearing in humans indicates that PP-IP signaling is important to hair cell maintenance and function within inner ear.
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http://dx.doi.org/10.1371/journal.pgen.1007297DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5891075PMC
March 2018

Inframe deletion of human is associated with deafness, vestibulopathy and vision impairment.

J Med Genet 2018 07 23;55(7):479-488. Epub 2018 Mar 23.

Department of Otorhinolaryngology Head and Neck Surgery, School of Medicine, University of Maryland, Baltimore, Maryland, USA.

Background: Usher syndrome (USH) is a neurosensory disorder characterised by deafness, variable vestibular areflexia and vision loss. The aim of the study was to identify the genetic defect in a Pakistani family (PKDF1051) segregating USH.

Methods: Genome-wide linkage analysis was performed by using an Illumina linkage array followed by Sanger and exome sequencing. Heterologous cells and mouse organ of Corti explant-based transfection assays were used for functional evaluations. Detailed clinical evaluations were performed to characterise the USH phenotype.

Results: Through homozygosity mapping, we genetically linked the USH phenotype segregating in family PKDF1051 to markers on chromosome 1p36.32-p36.22. The locus was designated . Using a combination of Sanger sequencing and exome sequencing, we identified a novel homozygous 18 base pair inframe deletion in Variants of , encoding the actin-bundling protein espin, have been previously associated with deafness and vestibular areflexia in humans with no apparent visual deficits. Our functional studies in heterologous cells and in mouse organ of Corti explant cultures revealed that the six deleted residues in affected individuals of family PKDF1051 are essential for the actin bundling function of espin demonstrated by ultracentrifugation actin binding and bundling assays. Funduscopic examination of the affected individuals of family PKDF1051 revealed irregular retinal contour, temporal flecks and disc pallor in both eyes. ERG revealed diminished rod photoreceptor function among affected individuals.

Conclusion: Our study uncovers an additional USH gene, assigns the USH1 phenotype to a variant of and provides a 12th molecular component to the USH proteome.
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http://dx.doi.org/10.1136/jmedgenet-2017-105221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6232856PMC
July 2018

Serum from CCl-induced acute rat injury model induces differentiation of ADSCs towards hepatic cells and reduces liver fibrosis.

Growth Factors 2017 10 7;35(4-5):144-160. Epub 2017 Nov 7.

a Centre of Excellence in Molecular Biology , University of Punjab , Lahore , Pakistan.

Cellular therapies hold promise to alleviate liver diseases. This study explored the potential of allogenic serum isolated from rat with acute CCl injury to differentiate adipose derived stem cells (ADSCs) towards hepatic lineage. Acute liver injury was induced by CCl which caused significant increase in serum levels of VEGF, SDF1α and EGF. ADSCs were preconditioned with 3% serum isolated from normal and acute liver injury models. ADSCs showed enhanced expression of hepatic markers (AFP, albumin, CK8 and CK19). These differentiated ADSCs were transplanted intra-hepatically in CCl-induced liver fibrosis model. After one month of transplantation, fibrosis and liver functions (alkaline phosphatase, ALAT and bilirubin) showed marked improvement in acute injury group. Elevated expression of hepatic (AFP, albumin, CK 18 and HNF4a) and pro survival markers (PCNA and VEGF) and improvement in liver architecture as deduced from results of alpha smooth muscle actin, Sirius red and Masson's trichome staining was observed.
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http://dx.doi.org/10.1080/08977194.2017.1392945DOI Listing
October 2017

Transplantation of stromal-derived factor 1α and basic fibroblast growth factor primed insulin-producing cells reverses hyperglycaemia in diabetic rats.

Growth Factors 2017 06 24;35(2-3):88-99. Epub 2017 Aug 24.

a Centre of Excellence in Molecular Biology , University of Punjab , Lahore , Pakistan.

The defective insulin production is associated with severely reduced islet cell mass leading to diabetes. Growth factors preconditioned stem cells have arisen as an effective therapy to treat many diseases including diabetes. The current study was designed to assess the effect of pretreatment of ASCs derived IPCs with combination of stromal cell derived factor 1 alpha (SDF1α) and basic fibroblast growth factor (bFGF) in improving glucose tolerance in streptozotocin induced diabetic rats. The results showed maximally significant reduction in hyperglycaemia and fibrosis, while up-regulation of survival and pancreas-specific genes, insulin levels and homing of transplanted cells in SDF-1α + bFGF IPCs transplanted rats as compared with other groups. Moreover, increased expression of insulin, glucagon and Glut-2 in pancreas of the SDF-1α + bFGF IPCs transplanted group indicated more regeneration of pancreas. Hence, the use of IPCs preconditioned with SDF-1α + bFGF would be more effective for treating diabetes.
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http://dx.doi.org/10.1080/08977194.2017.1363745DOI Listing
June 2017

A Systematic Review of End-of-Life Care Communication Skills Training for Generalist Palliative Care Providers: Research Quality and Reporting Guidance.

J Pain Symptom Manage 2017 09 4;54(3):417-425. Epub 2017 Aug 4.

Department of Palliative Care, Policy, and Rehabilitation, King's College London, Cicely Saunders Institute, London, UK; Population Health Sciences, University of Bristol, Bristol, United Kingdom.

Context: End-of-life care (EoLC) communication skills training for generalist palliative care providers is recommended in policy guidance globally. Although many training programs now exist, there has been no comprehensive evidence synthesis to inform future training delivery and evaluation.

Objectives: To identify and appraise how EoLC communication skills training interventions for generalist palliative care providers are developed, delivered, evaluated, and reported.

Methods: Systematic review. Ten electronic databases (inception to December 2015) and five relevant journals (January 2004 to December 2015) were searched. Studies testing the effectiveness of EoLC communication skills training for generalists were included. Two independent authors assessed study quality. Descriptive statistics and narrative synthesis are used to summarize the findings.

Results: From 11,441 unique records, 170 reports were identified (157 published, 13 unpublished), representing 160 evaluation studies of 153 training interventions. Of published papers, eight were of low quality, 108 medium, and 41 high. Few interventions were developed with service user involvement (n = 7), and most were taught using a mixture of didactics (n = 123), reflection and discussion (n = 105), and role play (n = 86). Evaluation designs were weak: <30% were controlled, <15% randomized participants. Over half (n = 85) relied on staff self-reported outcomes to assess effectiveness, and 49% did not cite psychometrically validated measures. Key information (e.g., training duration, participant flow) was poorly reported.

Conclusions: Despite a proliferation of EoLC communication skills training interventions in the literature, evidence is limited by poor reporting and weak methodology. Based on our findings, we present a CONSORT statement supplement to improve future reporting and encourage more rigorous testing.
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http://dx.doi.org/10.1016/j.jpainsymman.2017.04.008DOI Listing
September 2017

The Effect of Communication Skills Training for Generalist Palliative Care Providers on Patient-Reported Outcomes and Clinician Behaviors: A Systematic Review and Meta-analysis.

J Pain Symptom Manage 2017 09 1;54(3):404-416.e5. Epub 2017 Aug 1.

King's College London, Cicely Saunders Institute, London, United Kingdom.

Context: As most end-of-life care is provided by health care providers who are generalists rather than specialists in palliative care, effective communication skills training for generalists is essential.

Objectives: To determine the effect of communication training interventions for generalist palliative care providers on patient-reported outcomes and trainee behaviors.

Methods: Systematic review from searches of 10 databases to December 2015 (MEDLINE, EMBASE, PsycINFO, ERIC, CINAHL, CENTRAL, Web of Science, ICTRP, CORDIS, and OpenGrey) plus hand searching. Randomized controlled trials of training interventions intended to enhance generalists' communication skills in end-of-life care were included. Two authors independently assessed eligibility after screening, extracted data, and graded quality. Data were pooled for meta-analysis using a random-effects model. PRISMA guidelines were followed.

Results: Nineteen of 11,441 articles were eligible, representing 14 trials. Eleven were included in meta-analyses (patients n = 3144, trainees n = 791). Meta-analysis showed no effect on patient outcomes (standardized mean difference [SMD] = 0.10, 95% CI -0.05 to 0.24) and high levels of heterogeneity (chi-square = 21.32, degrees of freedom [df] = 7, P = 0.003; I = 67%). The effect on trainee behaviors in simulated interactions (SMD = 0.50, 95% CI 0.19-0.81) was greater than in real patient interactions (SMD = 0.21, 95% CI -0.01 to 0.43) with moderate heterogeneity (chi-square = 8.90, df = 5, P = 0.11; I = 44%; chi-square = 5.96, df = 3, P = 0.11; I = 50%, respectively). Two interventions with medium effects on showing empathy in real patient interactions included personalized feedback on recorded interactions.

Conclusions: The effect of communication skills training for generalists on patient-reported outcomes remains unclear. Training can improve clinicians' ability to show empathy and discuss emotions, at least in simulated consultations. Personalized feedback on recorded patient interactions may be beneficial.

Registration Number: CRD42014014777.
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http://dx.doi.org/10.1016/j.jpainsymman.2017.04.007DOI Listing
September 2017

Protective role of vitamin E preconditioning of human dermal fibroblasts against thermal stress in vitro.

Life Sci 2017 Sep 3;184:1-9. Epub 2017 Jul 3.

National Centre of Excellence in Molecular Biology, 87-West Canal Bank Road, University of the Punjab, Lahore, Pakistan; Allama Iqbal Medical College, University of Health Sciences, Lahore, Pakistan; Shaheed Zulfiqar Ali Bhutto Medical University (SZABMU), Islamabad, Pakistan. Electronic address:

Aims: Oxidative microenvironment of burnt skin restricts the outcome of cell based therapies of thermal skin injuries. The aim of this study was to precondition human dermal fibroblasts with an antioxidant such as vitamin E to improve their survival and therapeutic abilities in heat induced oxidative in vitro environment.

Main Methods: Fibroblasts were treated with 100μM vitamin E for 24h at 37°C followed by heat shock for 10min at 51°C in fresh serum free medium.

Key Findings: Preconditioning with vitamin E reduced cell injury as demonstrated by decreased expression of annexin-V, cytochrome p450 (CYP450) mediated oxidative reactions, senescence and release of lactate dehydrogenase (LDH) accomplished by down-regulated expression of pro-apoptotic BAX gene. Vitamin E preconditioned cells exhibited remarkable improvement in cell viability, release of paracrine factors such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), stromal derived factor-1alpha (SDF-1α) and also showed significantly up-regulated levels of PCNA, VEGF, BCL-XL, FGF7, FGF23, FLNβ and Col7α genes presumably through activation of phosphatidylinositol 3-kinase (PI3-K)/Akt pathway.

Significance: The results suggest that pretreatment of fibroblasts with vitamin E prior to transplantation in burnt skin speeds up the wound healing process by improving the antioxidant scavenging responses in oxidative environment of transplanted burn wounds.
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http://dx.doi.org/10.1016/j.lfs.2017.07.002DOI Listing
September 2017

Homozygosity Mapping and Genetic Analysis of Autosomal Recessive Retinal Dystrophies in 144 Consanguineous Pakistani Families.

Invest Ophthalmol Vis Sci 2017 04;58(4):2218-2238

Ophthalmic Genetics and Visual Function Branch, National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States.

Purpose: The Pakistan Punjab population has been a rich source for identifying genes causing or contributing to autosomal recessive retinal degenerations (arRD). This study was carried out to delineate the genetic architecture of arRD in the Pakistani population.

Methods: The genetic origin of arRD in a total of 144 families selected only for having consanguineous marriages and multiple members affected with arRD was examined. Of these, causative mutations had been identified in 62 families while only the locus had been identified for an additional 15. The remaining 67 families were subjected to homozygosity exclusion mapping by screening of closely flanking microsatellite markers at 180 known candidate genes/loci followed by sequencing of the candidate gene for pathogenic changes.

Results: Of these 67 families subjected to homozygosity mapping, 38 showed homozygosity for at least one of the 180 regions, and sequencing of the corresponding genes showed homozygous cosegregating mutations in 27 families. Overall, mutations were detected in approximately 61.8 % (89/144) of arRD families tested, with another 10.4% (15/144) being mapped to a locus but without a gene identified.

Conclusions: These results suggest the involvement of unmapped novel genes in the remaining 27.8% (40/144) of families. In addition, this study demonstrates that homozygosity mapping remains a powerful tool for identifying the genetic defect underlying genetically heterogeneous arRD disorders in consanguineous marriages for both research and clinical applications.
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http://dx.doi.org/10.1167/iovs.17-21424DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5397137PMC
April 2017

Molecular Genetic Analysis of Pakistani Families With Autosomal Recessive Congenital Cataracts by Homozygosity Screening.

Invest Ophthalmol Vis Sci 2017 04;58(4):2207-2217

Ophthalmic Genetics and Visual Function Branch, National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States.

Purpose: To identify the genetic origins of autosomal recessive congenital cataracts (arCC) in the Pakistani population.

Methods: Based on the hypothesis that most arCC patients in consanguineous families in the Punjab areas of Pakistan should be homozygous for causative mutations, affected individuals were screened for homozygosity of nearby highly informative microsatellite markers and then screened for pathogenic mutations by DNA sequencing. A total of 83 unmapped consanguineous families were screened for mutations in 33 known candidate genes.

Results: Patients in 32 arCC families were homozygous for markers near at least 1 of the 33 known CC genes. Sequencing the included genes revealed homozygous cosegregating sequence changes in 10 families, 2 of which had the same variation. These included five missense, one nonsense, two frame shift, and one splice site mutations, eight of which were novel, in EPHA2, FOXE3, FYCO1, TDRD7, MIP, GALK1, and CRYBA4.

Conclusions: The above results confirm the usefulness of homozygosity mapping for identifying genetic defects underlying autosomal recessive disorders in consanguineous families. In our ongoing study of arCC in Pakistan, including 83 arCC families that underwent homozygosity mapping, 3 mapped using genome-wide linkage analysis in unpublished data, and 30 previously reported families, mutations were detected in approximately 37.1% (43/116) of all families studied, suggesting that additional genes might be responsible in the remaining families. The most commonly mutated gene was FYCO1 (14%), followed by CRYBB3 (5.2%), GALK1 (3.5%), and EPHA2 (2.6%). This provides the first comprehensive description of the genetic architecture of arCC in the Pakistani population.
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http://dx.doi.org/10.1167/iovs.17-21469DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5397132PMC
April 2017