Whole genome sequencing followed by functional analysis of genomic deletion encompassing ERCC8 and NDUFAF2 genes in a non-consanguineous Indian family reveals dysfunctional mitochondrial bioenergetics leading to infant mortality.

Genomic investigations on an infant who presented with a putative mitochondrial disorder led to identification of compound heterozygous deletion with an overlapping region of ∼142 kb encompassing two nuclear encoded genes namely ERCC8 and NDUFAF2. Investigations on fetal-derived fibroblast culture demonstrated impaired bioenergetics and mitochondrial dysfunction, which explains the phenotype and observed infant mortality in the present study. The genetic findings from this study extended the utility of whole-genome sequencing as it led to development of a MLPA-based assay for carrier screening in the extended family and the prenatal testing aiding in the birth of two healthy children.

Biogenic Carbon Quantum Dots as a Neoteric Inducer in the Game of Directing Chondrogenesis.

The journey into the field of stem cell biology has been an endeavor of paramount advancement in biomedicine, establishing new horizons in the avenue of materiobiology. The creative drive of the scientific community focuses on ameliorating the utilization of stem cells, which is currently untapped on a large scale. With similar motivation, we present a nascent strategy of maneuvering biogenic carbon quantum dots (CQDs) to eclipse the toxic hurdles of chemical synthesis of carbon allotropes to serve as a biocompatible trident in stem cell biology employing a three-prong action of stem cell differentiation, imaging, and migration. The derivation of CQDs from garlic peels as a biogenic precursor abets in realizing the optophysical features of CQDs to image mesenchymal stem cells without hampering the biological systems with cytotoxicity. We report the versatility of biogenic CQDs to generate reactive oxygen species (ROS) to robustly influence stem cell migration and concomitantly chondrocyte differentiation from human Wharton's jelly mesenchymal stem cells (hWJ-MSCs). This was orchestrated without the use of chondrogenic induction factors, which was confirmed from the expression of chondrogenic markers (Col II, Col X, ACAN). Even the collagen content of cells incubated with CQDs was quite comparable with that of chondrocyte-induced cells. Thus, we empirically propose garlic peel-derived CQDs as a tangible advancement in stem cell biology from a materiobiological frame of reference to hone significant development in this arena.

Delineating the role of extracellular vesicles in cancer metastasis: A comprehensive review.

Extracellular vesicles (EVs) are subcellular messengers that aid in the formation and spread of cancer by enabling tumor-stroma communication. EVs develop from the very porous structure of late endosomes and hold information on both the intrinsic "status" of the cell and the extracellular signals absorbed by the cells from their surroundings. These EVs contain physiologically useful components, including as nucleic acids, lipids, and proteins, which have been found to activate important signaling pathways in tumor and tumor microenvironment (TME) cells, aggravating tumor growth. We highlight critical cell biology mechanisms that link EVS formation to cargo sorting in cancer cells in this review.Sorting out the signals that control EVs creation, cargo, and delivery will aid our understanding of carcinogenesis. Furthermore, we reviewed how cancer development and spreading behaviors are affected by coordinated communication between malignant and non-malignant cells. Herein, we studied the reciprocal exchanges via EVs in various cancer types. Further research into the pathophysiological functions of various EVs in tumor growth is likely to lead to the discovery of new biomarkers in liquid biopsy and the development of tumor-specific therapies.

Hypoxia preconditioning elicit differential response in tissue-specific MSCs via immunomodulation and exosomal secretion.

Mesenchymal stromal cells (MSCs) are emerging as an ideal candidate for regenerative medicine. It is known that the culture conditions impact the cellular properties of MSCs and their therapeutic behavior. Moreover, maintenance of MSCs in low oxygen tension for a short duration has shown to be beneficial for MSCs as it is similar to that of their physiological niche. However, the precise mechanism through which hypoxia pre-conditioning affects MSCs is not clear yet. Thus, in this study, we have investigated the effect of hypoxia exposure (1% O2) on tissue-specific MSCs over a period of time under serum-free culture conditions and evaluated the changes in expression of immuno-modulatory molecules and exosome biogenesis and secretion markers. It was observed that all MSCs responded differentially towards hypoxia exposure as indicated by the expression of HIF-1α. Moreover, this short-term exposure did not induce any changes in MSCs cellular morphology, proliferation rate, and surface marker profiling. In addition, we observed an enhancement in the expression of immunomodulatory factors (HLA-G, PGE-2, and IDO) after hypoxia exposure of 12 to 24 h in all tissue-specific MSCs. Interestingly, we have also observed the upregulation in exosome secretion that was further corelated to the upregulation of expression of exosome biogenesis and secretion markers (ALIX, TSG101, RAB27a, RAB27b). Though there was a differential response of MSCs where WJ-MSCs and BM-MSCs showed upregulation of these markers at 6-12 h of hypoxia pre-conditioning, while AD-MSCs showed similar changes beyond 24 h of hypoxia exposure.

Design of a phase III efficacy, immunogenicity, and safety study of 9-valent human papillomavirus vaccine in prevention of oral persistent infection in men.

BACKGROUND: Seven high-risk human papillomavirus (HPV) types (16/18/31/33/45/52/58) covered by the 9-valent HPV (9vHPV) vaccine cause >90% of HPV-related head and neck cancers (HNCs). An ongoing clinical trial (NCT04199689) was designed to evaluate 9vHPV vaccine efficacy against HPV oral persistent infection, a surrogate endpoint for HPV-related HNCs. METHODS: In this double-blind, placebo-controlled, international trial, men aged 20-45 years (N = 6000) are randomized 1:1 to receive 9vHPV vaccine or placebo on day 1, month 2, and month 6. The primary objective is to demonstrate whether 9vHPV vaccination reduces incidence of HPV16/18/31/33/45/52/58-related 6-month oral persistent infection. Incidence of HPV6/11-related 6-month oral persistent infection will be evaluated as a secondary endpoint. Oral rinse and gargle samples will be collected on day 1, month 7, month 12, and every 6 months thereafter for HPV detection by PCR. Primary analyses will be performed in per-protocol populations. Efficacy in this case-driven study will be analyzed upon accrual of ≥20 primary efficacy endpoint cases. Serum will be collected at day 1 and months 7, 12, 24, 36, and 42; anti-HPV antibody titers will be measured by competitive Luminex immunoassay. Data will be summarized as geometric mean titers and seropositivity rates. Injection-site and systemic adverse events (AEs) will be collected for 15 days post-any vaccination and serious AEs through 6 months after the last vaccination; deaths and vaccine-related serious AEs will be collected throughout the study. DISCUSSION: This trial is expected to generate important data regarding the potential for 9vHPV vaccine to prevent HPV-related head and neck disease.

Dexamethasone priming enhances stemness and immunomodulatory property of tissue-specific human mesenchymal stem cells.

BACKGROUND: Human Mesenchymal Stem Cells (hMSCs) represent a promising cell source for cell-based therapy in autoimmune diseases and other degenerative disorders due to their immunosuppressive, anti-inflammatory and regenerative potentials. Belonging to a glucocorticoid family, Dexamethasone (Dex) is a powerful anti-inflammatory compound that is widely used as therapy in autoimmune disease conditions or allogeneic transplantation. However, minimal immunomodulatory effect of hMSCs may limit their therapeutic uses. Moreover, the effect of glucocorticoids on the immunomodulatory molecules or other regenerative properties of tissue-specific hMSCs remains unknown. METHOD: Herein, we evaluated the in vitro effect of Dex at various dose concentrations and time intervals, 1000 ng/ml, 2000 ng/ml, 3000 ng/ml and 24 h, 48 h respectively, on the basic characteristics and immunomodulatory properties of Bone marrow derived MSC (BM-MSCs), Adipose tissue derived MSCs (AD-MSCs), Dental Pulp derived MSC (DP-MSCs) and Umbilical cord derived MSCs (UC-MSCs). RESULTS: The present study indicated that the concentration of Dex did not ramify the cellular morphology nor showed cytotoxicity as well as conserved the basic characteristics of tissue specific hMSCs including cell proliferation and surface marker profiling. However, quite interestingly it was observed that the stemness markers (Oct-4, Sox-2, Nanog and Klf-4) showed a significant upregulation in DP-MSCs and AD-MSCs followed by UC-MSCs and BM-MSCs. Additionally, immunomodulatory molecules, Prostaglandin E-2 (PGE-2), Indoleamine- 2,3-dioxygenase (IDO) and Human Leukocyte Antigen-G (HLA-G) were seen to be upregulated in a dose-dependent manner. Moreover, there was a differential response of tissue specific hMSCs after pre-conditioning with Dex during mixed lymphocyte reaction, wherein UC-MSCs and DP-MSCs showed enhanced immunosuppression as compared to AD-MSCs and BM-MSCs, thereby proving to be a better candidate for therapeutic applications in immune-related diseases. CONCLUSION: Dex preconditioning improved the hMSCs immunomodulatory property and may have reduced the challenge associated with minimal potency and strengthen their therapeutic efficacy. Preconditioning of tissue specific hMSCs with dexamethasone biomanufacturers the enhanced potential hMSCs with better stemness and immunomodulatory properties for future therapeutics.

Graphene nanofiber composites for enhanced neuronal differentiation of human mesenchymal stem cells.

Aim: To differentiate mesenchymal stem cells into functional dopaminergic neurons using an electrospun polycaprolactone (PCL) and graphene (G) nanocomposite. Methods: A one-step approach was used to electrospin the PCL nanocomposite, with varying G concentrations, followed by evaluating their biocompatibility and neuronal differentiation. Results: PCL with exiguous graphene demonstrated an ideal nanotopography with an unprecedented combination of guidance stimuli and substrate cues, aiding the enhanced differentiation of mesenchymal stem cells into dopaminergic neurons. These newly differentiated neurons were seen to exhibit unique neuronal arborization, enhanced intracellular Ca2+ influx and dopamine secretion. Conclusion: Having cost-effective fabrication and room-temperature storage, the PCL-G nanocomposites could pave the way for enhanced neuronal differentiation, thereby opening a new horizon for an array of applications in neural regenerative medicine.

Human Acquired Aplastic Anemia Patients' Bone-Marrow-Derived Mesenchymal Stem Cells Are Not Influenced by Hematopoietic Compartment and Maintain Stemness and Immune Properties.

METHODS: In the current study, we investigated the morphological differences, proliferation capacity, population doubling time (PDT), surface marker profiling, trilineage differentiation potential, and immunosuppressive ability of BM Mesenchymal Stem Cells (BM-MSCs) from untreated aAA patients and in the same number of age- and gender-matched controls. RESULTS: We observed similar morphology, proliferation capacity, phenotype, trilineage differentiation potential, and immunomodulatory properties of BM-MSCs in aAA patients and control subjects. CONCLUSION: Our results confirm that the basic and immunosuppressive properties of BM-MSCs from aAA patients do not differ from normal BM-MSCs. Our data suggest that BM-MSCs from aAA patients might not be involved in disease pathogenesis. However, owing to a smaller number of samples, it is not conclusive, and future studies with more exhaustive investigation at transcriptome level are warranted.

Immunogenicity and safety of a nine-valent human papillomavirus vaccine in women 27-45 years of age compared to women 16-26 years of age: An open-label phase 3 study.

BACKGROUND: Efficacy of the nine-valent human papillomavirus (9vHPV; HPV types 6/11/16/18/31/33/45/52/58) vaccine was demonstrated in a phase 3 study in women 16-26 years of age. We present a phase 3 immunogenicity and safety study of the 9vHPV vaccine in women 27-45 versus 16-26 years of age. METHODS: This international, open-label study (NCT03158220) was conducted in women 16-45 years of age. Participants (16-26 years, n = 570 and 27-45 years, n = 642) received a three-dose 9vHPV vaccination regimen (day 1, month 2, month 6). Month 7 geometric mean titers (GMTs) and seroconversion percentages to anti-HPV 6/11/16/18/31/33/45/52/58 were assessed. Participants were followed for safety throughout the study. RESULTS: At month 7, anti-HPV 6/11/16/18/31/33/45/52/58 GMTs in women 27-45 years were compared to those in women 16-26 years of age. The primary hypothesis of non-inferiority of anti-HPV 16/18/31/33/45/52/58 GMTs in older versus younger women was met. The lower bound of the GMT ratio 95% confidence interval (27-45 years to 16-26 years) was 0.60-0.67 depending on HPV type, exceeding the non-inferiority margin of 0.5 for all HPV types. Month 7 seroconversion percentages in women 27-45 years of age were >99% for all HPV types. Injection-site and vaccine-related systemic adverse events (AEs) were observed in 87.5% and 25.1% of women 16-26 years, and 85.2% and 24.1% of women 27-45 years of age, respectively; no vaccine-related serious AEs were reported and no deaths occurred during the study. CONCLUSIONS: The 9vHPV vaccine elicited non-inferior anti-HPV GMTs in women 27-45 years compared with women 16-26 years of age for HPV 16/18/31/33/45/52/58. The vaccine was generally well tolerated with a similar AE profile across the age groups. These data support bridging 9vHPV vaccine efficacy findings in women 16-26 years to women 27-45 years of age. Clinical trial registration NCT03158220.

Influence of varying concentrations of chitosan coating on the pore wall of polycaprolactone based porous scaffolds for tissue engineering application.

The study's purpose was to fabricate a 3-D porous scaffold, in which chitosan was coated onto the pore wall of polycaprolactone (PCL) scaffolds as a bioactive agent to maximize the cell recognition signals, to improve the osteoconductivity of the scaffolds. The pppporogen leaching technique has been modified and used in the fabrication process, comprising of the coating of chitosan over the porogen followed by transferring of coating to the pore wall of the PCL scaffold. The cytotoxicity and hemolysis results indicated chitosan's presence over the surface of the scaffold's pore walls has significantly enhanced its biocompatibility. Scaffolds coated with 2.5 %(w/v) chitosan shows 6.74 % increase in porosity and 207.96 % upsurge in mechanical strength, compared to PCL scaffolds. The Gene-expression also proves the study groups of scaffolds show the minimal osteogenic expression. Therefore, chitosan coating over the scaffold's pore wall's surface opens an unconventional approach for tissue engineering applications.

Tissue-specific mesenchymal stem cell-dependent osteogenesis in highly porous chitosan-based bone analogs.

Among conventional fabrication techniques, freeze-drying process has widely been investigated for polymeric implants. However, the understanding of the stem cell progenitor-dependent cell functionality modulation and quantitative analysis of early osseointegration of highly porous scaffolds have not been explored. Here, we developed a novel, highly porous, multimaterial composite, chitosan/hydroxyapatite/polycaprolactone (CHT/HA/PCL). The in vitro studies have been performed using mesenchymal stem cells (MSCs) from three tissue sources: human bone marrow-derived MSCs (BM-MSCs), adipose-derived MSCs (AD-MSCs), and Wharton's jelly-derived MSCs (WJ-MSCs). Although cell attachment and metabolic activity [3-4,5-dimethylthiazol-2yl-(2,5 diphenyl-2H-tetrazoliumbromide) assay] were ore enhanced in WJ-MSC-laden CHT/HA/PCL composites, scanning electron microscopy, real-time gene expression (alkaline phosphatase [ALP], collagen type I [Col I], osteocalcin [OCN], and bone morphogenetic protein 4 [BMP-4]), and immunostaining (COL I, ß-CATENIN, OCN, and SCLEROSTIN [SOST]) demonstrated pronounced osteogenesis with terminal differentiation on BM-MSC-laden CHT/HA/PCL composites only. The enhanced cell functionality on CHT/HA/PCL composites was explained in terms of interplay among the surface properties and the optimal source of MSCs. In addition, osteogenesis in rat tibial model over 6 weeks confirmed a better ratio of bone volume to the total volume for BM-MSC-laden composites over scaffold-only and defect-only groups. The clinically conformant combination of 3D porous architecture with pore sizes varying in the range of 20 to 200 µm together with controlled in vitro degradation and early osseointegration establish the potential of CHT/HA/PCL composite as a potential cancellous bone analog.

Efficient Labeling of Human Mesenchymal Stem Cells Using Iron Oxide Nanoparticles.

Stem cells have been used in multiple clinical trials. Tracking these transplanted cells in vivo will provide real-time information on the fate of these cells. Iron oxide labeling is one such uncomplicated noninvasive labeling method. These transformed nanocrystals can be used for varied applications including stem-cell tracking, magnetic resonance imaging, and theranostics. Here we elucidate the protocol for iron oxide nanoparticles synthesis (IONPS) and labeling of mesenchymal stem cells which can be used for imaging and tracking cells to understand their fate in in vivo studies.

An improvised one-step sucrose cushion ultracentrifugation method for exosome isolation from culture supernatants of mesenchymal stem cells.

BACKGROUND: Exosomes are nanovesicles (30-120 nm) of endosomal origin. These exosomes contain various functional proteins and RNAs that could be used for therapeutic purposes. Currently, having a standard method for exosome isolation retaining its biological properties with increased yield and purity is a major challenge. The most commonly used method is differential ultracentrifugation but it has its own disadvantages, which include high time consumption, low yield due to disruption of exosome integrity, and high protein contaminants. In this study, we have identified an improved method addressing these problems for exosome isolation using ultracentrifugation since it is cost-effective and used worldwide. METHOD: We have compared differential ultracentrifugation with the modified method called one-step sucrose cushion ultracentrifugation for exosome isolation. The conditioned serum-free media from human mesenchymal stem cells cultured for 48 h was collected for exosome isolation. The cellular debris was removed by centrifugation at 300g for 10 min, followed by centrifugation at 10,000g for 30 min to remove microvesicles. Equal volumes of pre-processed conditioned media were used for exosome isolation by direct ultracentrifugation and one-step sucrose cushion ultracentrifugation. The exosomes isolated using these methods were characterized for their size, morphology, concentration, and surface marker protein expression. RESULT: It was observed that the recovery of exosomes with cup-shaped morphology from one-step sucrose cushion ultracentrifugation was comparatively high as estimated by nanoparticle tracking analysis and electron microscopy. These results were confirmed by Western blotting and flow cytometry. CONCLUSION: We conclude that this one-step sucrose cushion ultracentrifugation method provides an effective and reproducible potential standard method which could be used for various starting materials for isolating exosomes. We believe that this method will have a wide application in the field of extracellular vesicle research where exosome isolation with high yield and purity is an imperative step. Schematic representation of comparison of UC and SUC exosome isolation methods for tissue-specific human mesenchymal stem cells. The SUC isolation method yields a greater number of cup-shaped exosomes with a relatively homogenous population for mass-scale production of exosomes for downstream analysis. ABBREVIATIONS: SUC One-step sucrose cushion ultracentrifugation, UC Direct ultracentrifugation.

H-ras Inhibits the Hippo Pathway by Promoting Mst1/Mst2 Heterodimerization.

The protein kinases Mst1 and Mst2 have tumor suppressor activity, but their mode of regulation is not well established. Mst1 and Mst2 are broadly expressed and may have certain overlapping functions in mammals, as deletions of both Mst1 and Mst2 together are required for tumorigenesis in mouse models [1-3]. These kinases act via a three-component signaling cascade comprising Mst1 and Mst2, the protein kinases Lats1 and Lats2, and the transcriptional coactivators Yap and Taz [4-6]. Mst1 and Mst2 contain C-terminal SARAH domains that mediate their homodimerization as well as heterodimerization with other SARAH domain-containing proteins, which may regulate Mst1/Mst2 activity. Here we show that, in addition to forming homodimers, Mst1 and Mst2 heterodimerize in cells, this interaction is mediated by their SARAH domains and is favored over homodimers, and these heterodimers have much-reduced protein kinase activity compared to Mst1 or Mst2 homodimers. Mst1/Mst2 heterodimerization is strongly promoted by oncogenic H-ras, and this effect requires activation of the Erk pathway. Cells lacking Mst1, in which Mst1/Mst2 heterodimers are not possible, are resistant to H-ras-mediated transformation and maintain active hippo pathway signaling compared to wild-type cells or cells lacking both Mst1 and Mst2. Our results suggest that H-ras, via an Erk-dependent mechanism, downregulates Mst1/Mst2 activity by inducing the formation of inactive Mst1/Mst2 heterodimers.

Regulation of mammalian Ste20 (Mst) kinases.

Initially identified as mammalian homologs to yeast Ste20 kinases, the mammalian sterile twenty-like (Mst) 1/2 kinases have been widely investigated subsequent to their rediscovery as key components of the Hippo tumor suppressor pathway in flies. To date, our understanding of Mst substrates and downstream signaling outstrips our knowledge of how these enzymes are controlled by upstream signals. While much remains to be discovered regarding the mechanisms of Mst regulation, it is clear that Mst1 kinase activity is governed at least in part by its state of dimerization, including self-association and also heterodimerization with various other signaling partners. Here we review the basic architecture of Mst signaling and function and discuss recent advances in our understanding of how these important kinases are regulated.

Molecular pathways: targeting the kinase effectors of RHO-family GTPases.

RHO GTPases, members of the RAS superfamily of small GTPases, are adhesion and growth factor-activated molecular switches that play important roles in tumor development and progression. When activated, RHO-family GTPases such as RAC1, CDC42, and RHOA, transmit signals by recruiting a variety of effector proteins, including the protein kinases PAK, ACK, MLK, MRCK, and ROCK. Genetically induced loss of RHO function impedes transformation by a number of oncogenic stimuli, leading to an interest in developing small-molecule inhibitors that either target RHO GTPases directly, or that target their downstream protein kinase effectors. Although inhibitors of RHO GTPases and their downstream signaling kinases have not yet been widely adopted for clinical use, their potential value as cancer therapeutics continues to facilitate pharmaceutical research and development and is a promising therapeutic strategy.

ArhGAP15, a Rac-specific GTPase-activating protein, plays a dual role in inhibiting small GTPase signaling.

Signaling from small GTPases is a tightly regulated process. In this work we used a protein microarray screen to identify the Rac-specific GAP, ArhGAP15, as a substrate of the Rac effectors Pak1 and Pak2. In addition to serving as a substrate of Pak1/2, we found that ArhGAP15, via its PH domain, bound to these kinases. The association of ArhGAP15 to Pak1/2 resulted in mutual inhibition of GAP and kinase catalytic activity, respectively. Knock-down of ArhGAP15 resulted in activation of Pak1/2, both indirectly, as a result of Rac activation, and directly, as a result of disruption of the ArhGAP15/Pak complex. Our data suggest that ArhGAP15 plays a dual negative role in regulating small GTPase signaling, by acting at the level of the GTPase itself, as well interacting with its effector, Pak kinase.

The tumor suppressor Mst1 promotes changes in the cellular redox state by phosphorylation and inactivation of peroxiredoxin-1 protein.

The serine/threonine protein kinases Mst1 and Mst2 can be activated by cellular stressors including hydrogen peroxide. Using two independent protein interaction screens, we show that these kinases associate, in an oxidation-dependent manner, with Prdx1, an enzyme that regulates the cellular redox state by reducing hydrogen peroxide to water and oxygen. Mst1 inactivates Prdx1 by phosphorylating it at Thr-90 and Thr-183, leading to accumulation of hydrogen peroxide in cells. These results suggest that hydrogen peroxide-stimulated Mst1 activates a positive feedback loop to sustain an oxidizing cellular state.