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Immune dysfunction is critical in pathogenesis of cutaneous T-cell lymphoma (CTCL). Few studies have reported abnormal cytokine profile and dysregulated T-cell functions during the onset and progression of certain types of lymphoma. However, the presence of IL9-producing Th9 cells and their role in tumor cell metabolism and survival remain unexplored. With this clinical study, we performed multidimensional blood endotyping of CTCL patients before and after standard photo/chemotherapy and revealed distinct immune hallmarks of the disease. Importantly, there was a higher frequency of “skin homing” Th9 cells in CTCL patients with early (T1 and T2) and advanced-stage disease (T3 and T4). However, advanced-stage CTCL patients had severely impaired frequency of skin-homing Th1 and Th17 cells, indicating attenuated immunity. Treatment of CTCL patients with standard photo/chemotherapy decreased the skin-homing Th9 cells and increased the Th1 and Th17 cells. Interestingly, T cells of CTCL patients express IL9 receptor (IL9R), and there was negligible IL9R expression on T cells of healthy donors. Mechanistically, IL9/IL9R interaction on CD3+ T cells of CTCL patients and Jurkat cells reduced oxidative stress, lactic acidosis, and apoptosis and ultimately increased their survival. In conclusion, coexpression of IL9 and IL9R on T cells in CTCL patients indicates the autocrine-positive feedback loop of Th9 axis in promoting the survival of malignant T cells by reducing the oxidative stress.Implications:The critical role of Th9 axis in CTCL pathogenesis indicates that strategies targeting Th9 cells might harbor significant potential in developing robust CTCL therapy.

Supplementary table S1: Follow-up patient's clinical details

This study visualizes crowdfunding as a strategically important tool for the revival and survival of watermills in Uttarakhand. There are 500,000 watermills in the region from Afghanistan to Myanmar, between 100,000 to 200,000 in India, and 60,000 in Uttarakhand. These watermills have the potential to generate electricity up to 65000 MW at the rate of 2500 MW/hr that could be monetized at $ 17 million per hour in the northern mountain ranges. These watermills are also used for grinding purposes leading to multidimensional ways for sustainable livelihood. These watermills contribute to the social capital of the region immensely. The Government of India has supported these traditional knowledge-based activities by providing capital support and fiscal incentives. It is observed that despite the availability of government funds, the water mill owners lack finance for revenue expenditure, which is one of the significant barriers to the sustenance of water mills. Repair and maintenance of watermills, support to scale-up activities and other marketing activities, etc. are examples of recurring revenue expenditure. The goal of this descriptive paper broader view of the concept and evolution of crowdfunding based on a bibliometric tool. It analyses all crowdfunding-related publications in Scopus-indexed journals. It analyses the parameters number of papers published during 2011-2021based on various parameters. This study identifies the donation-based method as the least researched method of crowdfunding. It is also brought out that the donation-based method will not be useful in the case of repetitive crowdfunding initiatives.

Fibrosis, the pathological end stage of chronic inflammatory diseases, results from extracellular matrix deposition by fibrogenic fibroblasts. In this issue of Cell Stem Cell, Sobecki et al. (2022) develop a novel vaccination-based immunotherapy against fibrogenic progenitor-restricted antigens, leading to the regression of fibrosis in concert with liver and lung regeneration.

Online/blended teaching and learning practices are being implemented worldwide and gaining adoption among the students over the years to meet the demands of the changing environment. However, there is a need to understand whether the students are responding to this change and what their perception towards online/blended courses is. This study attempts to understand the student experiences, both good and bad, in blended/online learning and the components and activities they expect in an online learning environment. The study identifies critical success factors of effective online environment in higher education institutions and universities. To measure student experience and satisfaction, primary data of 1649 students were collected from different schools in higher education. The results of the study can be used by academics, corporate, and consumer fields to introduce innovative content and delivery strategies towards a student-centric approach, which would further increase student acceptance of online learning environments.

The coronavirus disease originated from the city of Wuhan in late 2019. Colleges and schools faced various challenges, but they accepted the fact and soon went online and learning continued. This study makes an attempt to understand student behaviour towards the online learning environment and to measure the relationship between the various online learning environment factors and student behaviour. Primary data of 500 students were collected using stratified random sampling from different schools and universities with the help of a questionnaire. Data were analysed using Karl Pearson correlation. Results show a weak correlation between online learning factors and student behaviour towards learning. The exclusivity of this study is that it has attempted to fill the wide gaps existing in the field of online learning. This research work confirms the importance of student preferences in analysing and measuring the experiences of students and identifies their significant effect in online learning especially in higher education.

The engagement of students and faculty in the teaching and learning process of Higher Education is key to ensure the career readiness of graduates, and their future ability to make a positive impact in their communities and the world. It is in this context that Positive Leadership has been piloted in a young, comprehensive Indian university, where the two professors that led this innovative approach were trained so that the delivery of their subjects enhanced the principles of positive meaning, positive climate, positive communication and positive relationships. The research carried out during this pilot study incorporated a mixed-methods approach, which allowed to scientifically assess the very positive impact of the initiative. Both students and faculty appreciated a more human methodology and reported highly satisfactory results, including boosted engagement and improved grades.

High numbers of autologous human primary keratinocytes (HPKs) are required for patients with burns, wounds and for gene therapy of skin disorders., High numbers of autologous human primary keratinocytes (HPKs) are required for patients with burns, wounds and for gene therapy of skin disorders. Although freshly isolated HPKs exhibit a robust regenerative capacity, traditional methodology fails to provide a sufficient number of cells. Here we demonstrated a well characterized, non-cytotoxic and inert hydrogel as a substrate that mimics skin elasticity, which can accelerate proliferation and generate higher numbers of HPKs compared to existing tissue culture plastic (TCP) dishes. More importantly, this novel method was independent of feeder layer or any exogenous pharmaceutical drug. The HPKs from the hydrogel-substrate were functional as demonstrated by wound-healing assay, and the expression of IFN-γ-responsive genes (CXCL10, HLADR). Importantly, gene delivery efficiency by a lentiviral based delivery system was significantly higher in HPKs cultured on hydrogels compared with TCP. In conclusion, our study provides the first evidence that cell-material mechanical interaction is enough to provide a rapid expansion of functional keratinocytes that might be used as autologous grafts for skin disorders.

Recent studies have described the remarkable clinical outcome of anti-CD19 chimeric antigen receptor (CAR) T cells in treating B-cell malignancies. However, over 50% of patients develop life-threatening toxicities associated with cytokine release syndrome which may limit its utilization in low-resource settings. To mitigate the toxicity, we designed a novel humanized anti-CD19 CAR T cells by humanizing the framework region of single-chain variable fragment (scFv) derived from a murine FMC63 mAb and combining it with CD8α transmembrane domain, 4-1BB costimulatory domain, and CD3ζ signaling domain (h1CAR19-8BBζ). Docking studies followed by molecular dynamics simulation revealed that the humanized anti-CD19 scFv (h1CAR19) establishes higher binding affinity and has a flexible molecular structure with CD19 antigen compared with murine scFv (mCAR19). Ex vivo studies with CAR T cells generated from healthy donors and patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL) expressing either h1CAR19 or mCAR19 showed comparable antitumor activity and proliferation. More importantly, h1CAR19-8BBζ T cells produced lower levels of cytokines (IFNγ, TNFα) upon antigen encounter and reduced the induction of IL6 cytokine from monocytes than mCAR19-8BBζ T cells. There was a comparable proliferation of h1CAR19-8BBζ T cells and mCAR19-8BBζ T cells upon repeated antigen encounter. Finally, h1CAR19-8BBζ T cells efficiently eliminated NALM6 tumor cells in a preclinical model. In conclusion, the distinct structural modification in CAR design confers the novel humanized anti-CD19 CAR with a favorable balance of efficacy to toxicity providing a rationale to test this construct in a phase I trial.

IL-9 is produced by Th9 cells and is classically known as a growth-promoting cytokine. Although protumorigenic functions of IL-9 are described in T cell lymphoma, recently, we and others have reported anti-tumor activities of IL-9 in melanoma mediated by mast cells and CD8+ T cells. However, involvement of IL-9 in invasive breast and cervical cancer remains unexplored. In this study, we demonstrate IL-9–dependent inhibition of metastasis of both human breast (MDA-MB-231 and MCF-7) and cervical (HeLa) tumor cells in physiological three-dimensional invasion assays. To dissect underlying mechanisms of IL-9–mediated suppression of invasion, we analyzed IL-9–dependent pathways of cancer cell metastasis, including proteolysis, contractility, and focal adhesion dynamics. IL-9 markedly blocked tumor cell–collagen degradation, highlighting the effects of IL-9 on extracellular matrix remodeling. Moreover, IL-9 significantly reduced phosphorylation of myosin L chain and resultant actomyosin contractility and also increased focal adhesion formation. Finally, IL-9 suppressed IL-17– and IFN-γ–induced metastasis of both human breast (MDA-MB-231) and cervical (HeLa) cancer cells. In conclusion, IL-9 inhibits the metastatic potential of breast and cervical cancer cells by controlling extracellular matrix remodeling and cellular contractility.

Online learning has become one of the fastest moving trend and is an effective alternative to the traditional classroom learning. It aims to provide a configurable infrastructure which collaborates learning content, technology and delivery into a single solution to move society ahead by imparting education quickly, effectively, and economically. This study seeks to delve into this aspect and attempts to understand the students perspective of an effective online delivery of academic content to undergraduate and postgraduate students. This study is exploratory in nature. The objectives of the study are to understand the students experience both good and bad in online learning, the knowledge, skills abilities they expect from online instructors and the components and activities they expect in an online learning environment. The results of the study can be used for providing relevant training to the prospective and aspiring online teachers and thus contributing to excellence in online teaching in higher education.

The present paper aims at analysing the time investment involved in delivering blended and online courses and assess the increasing work load of faculty in online and blended teaching environment. For the study, twenty two faculty members from different departments agreed to log their daily activities related to their respective online and blended courses. The log diary data which was collected over a period of one semester shows individual variation in the performance of different activities by the instructor. However, it also reflects considerable increase in the time spent by the instructors in delivering online/blended courses. This time is over and above the contact hours scheduled for the class. Analyzing the time spent in different activities, clearly shows that developing, delivering and administration of online sessions increases the workload of instructors. Inferences are also drawn regarding the changed skill set required for delivering online courses as well as the changed expectations of the online students. The paper suggests the need to review and rationalize the work load of faculty members delivering online courses.

Immune dysfunction is critical in pathogenesis of cutaneous T-cell lymphoma (CTCL). Few studies have reported abnormal cytokine profile and dysregulated T-cell functions during the onset and progression of certain types of lymphoma. However, the presence of IL9-producing Th9 cells and their role in tumor cell metabolism and survival remain unexplored. With this clinical study, we performed multidimensional blood endotyping of CTCL patients before and after standard photo/chemotherapy and revealed distinct immune hallmarks of the disease. Importantly, there was a higher frequency of “skin homing” Th9 cells in CTCL patients with early (T1 and T2) and advanced-stage disease (T3 and T4). However, advanced-stage CTCL patients had severely impaired frequency of skin-homing Th1 and Th17 cells, indicating attenuated immunity. Treatment of CTCL patients with standard photo/chemotherapy decreased the skin-homing Th9 cells and increased the Th1 and Th17 cells. Interestingly, T cells of CTCL patients express IL9 receptor (IL9R), and there was negligible IL9R expression on T cells of healthy donors. Mechanistically, IL9/IL9R interaction on CD3+ T cells of CTCL patients and Jurkat cells reduced oxidative stress, lactic acidosis, and apoptosis and ultimately increased their survival. In conclusion, coexpression of IL9 and IL9R on T cells in CTCL patients indicates the autocrine-positive feedback loop of Th9 axis in promoting the survival of malignant T cells by reducing the oxidative stress. Implications: The critical role of Th9 axis in CTCL pathogenesis indicates that strategies targeting Th9 cells might harbor significant potential in developing robust CTCL therapy.

Lymphocytes especially autologous T cells have been used for the treatment of numerous indications including cancers, autoimmune disorders and infectious diseases. Very recently, FDA approved Chimeric Antigen Receptor T cells (CAR T cells) therapy for relapse and refractory CD19+ B cell acute lymphoblastic leukemia (r/r B-ALL) and r/r diffuse large B cell lymphoma (r/r DLBCL) upon their remarkable success in multiple Phase I-II clinical trials. While CAR T cells are considered as major breakthrough in the field of cancer immunotherapy, the regulation of CAR T cells remains poorly understood. In this review we will discuss the strategies that regulate the CAR T cells efficacy and persistence with focus on roles of different structural component of CAR construct. Different domains of CAR construct, for example, antigen binding domain, hinge, transmembrane, and signaling domain as well as immune-regulatory cytokines have significant impact on CAR T cell efficacy. Finally, this review will highlight the strategies that will promote CAR T cells efficacy and will reduce the toxicity.

Forest fires due to pine needles in the northwestern Himalayas during summers are a very common phenomenon every year causing major harm to flora and fauna. They are a major contributor to climate change as forest fires destroy hectares of land covered by forest and emit greenhouse gases in the atmosphere. One possible solution to this problem is to generate energy from the bio residue of pine tree. This paper intends to identify, categorize and prioritize the major factors that hinders and are barriers to energy generation using pine needles in the state of Uttarakhand, India. This paper adopts a multi-criteria decision-making approach based on the technique of fuzzy Analytical Hierarchy Process. Sensitivity analysis is also used to test the robustness of this technique. The study finds that Legal and regulatory factors are the most critical and categorized as major influences of such renewable energy generation project, which includes government policy, project guidelines, financial support, technical support, and government clearances. The second barrier is economic which majorly includes cost factor associated with the development and sustenance of such energy generation projects. Technological and market related factors are next on the priority to energy generation using pine needles through limitations such as topography, hilly terrain, seasonal availability, supply chain, poor marketing. Human resource barriers stands at the fifth place and has less influence on such energy generation initiatives. This paper will help the policy makers to understand the priorities while solving energy generation issues to reduce forest fires and contribute to mitigate climate change.

T cells mediate skin immune surveillance by secreting specific cytokines and regulate numerous functions of keratinocytes, including migration during homeostasis and disease pathogenesis. Keratinocyte migration is mediated mainly by proteolytic cleavage of the extracellular matrix and/or by cytoskeleton reorganization. However, the cross-talk between T cell cytokines and actomyosin machinery of human primary keratinocytes (HPKs), which is required for cytoskeleton reorganization and subsequent migration, remains poorly examined. In this study, we describe that IL-9 profoundly reduced the actin stress fibers, inhibited contractility, and reduced the cortical stiffness of HPKs, which resulted in inhibition of the migration potential of HPKs in an adhesion- and MMP-independent manner. Similarly, IL-9 inhibited the IFN-γ–induced migration of HPKs by inhibiting the actomyosin machinery (actin stress fibers, contractility, and stiffness). IL-17A increased the actin stress fibers, promoted cellular contractility, and increased proteolytic collagen degradation, resulting in increased migration potential of HPKs. However, IL-9 inhibited the IL-17A–mediated HPKs migration. Mechanistically, IL-9 inhibited the IFN-γ– and IL-17A–induced phosphorylation of myosin L chain in HPKs, which is a major regulator of the actomyosin cytoskeleton. Finally, in addition to HPKs, IL-9 inhibited the migration of A-431 cells (epidermoid carcinoma cells) induced either by IFN-γ or IL-17A. In conclusion, our data demonstrate the influence of T cell cytokines in differentially regulating the actomyosin cytoskeleton and migration potential of human keratinocytes, which may have critical roles in skin homeostasis and pathogenesis of inflammatory diseases as well as skin malignancies.

During this Information age we witness an ever increasing use of Information and Communication Technology (ICT) in education space. The present paper describes how a University attempted to digitize its teaching and learning processes. It improvised and strengthened its already available IT infrastructure in order to support e-learning. Dedicated teams were formed and trained to develop small learning modules in house for certain subjects. These modules were then made available to a few batches of undergraduate and post graduate students pursuing engineering and management courses. These online modules were delivered in addition to the face-to-face lectures by the faculty members teaching those seven subjects. This exercise was run for two semesters and continuous feedback was taken from the students and the instructors. This paper documents the important learnings from this experiment along with the challenges faced and critical success factors identified in the process.

T cell therapy has shown remarkable promise in multiple malignancies including melanoma and Acute Lymphoblastic Leukemia (ALL). Recent data demonstrated the differential efficacy of various subsets of T-helper cells in tumor regression. Th9 cells, the new member of T helper cell family, possess superior anti-tumor activity compared to Th1 and Th2 cells in murine model of melanoma. Therefore, it is important to examine the anti-tumor activity of specific subsets of Th cells in tumor models. Here, we describe the methodology of examining the anti-tumor activity of Th9 cells in murine model of melanoma.

T cell therapy has shown remarkable promise in multiple malignancies including melanoma and Acute Lymphoblastic Leukemia (ALL). Recent data demonstrated the differential efficacy of various subsets of T-helper cells in tumor regression. Th9 cells, the new member of T helper cell family, possess superior anti-tumor activity compared to Th1 and Th2 cells in murine model of melanoma. Therefore, it is important to examine the anti-tumor activity of specific subsets of Th cells in tumor models. Here, we describe the methodology of examining the anti-tumor activity of Th9 cells in murine model of melanoma.

Background Blepharophimosis-ptosis-epicanthus inversus syndrome is an autosomal dominant condition because of mutations or deletions of the FOXL2 gene. Microcephaly is not associated with FOXL2 mutations but has been reported in individuals with chromosome 3q deletions, which include the FOXL2 gene and other contiguous genes. The ATR gene has been reported as a candidate gene for microcephaly in individuals with contiguous deletion of chromosome 3q involving the FOXL2 gene. Patient We describe a girl with blepharophimosis-ptosis-epicanthus inversus syndrome along with acquired microcephaly and intellectual disability. Results Our patient had a deletion of chromosome 3q22.2q23, which does not include the ATR gene but does include the PIK3CB gene as a candidate gene for microcephaly. Conclusion We propose that the PIK3CB gene included in our patient's chromosome 3q deletion may be the gene responsible for microcephaly and other patients with blepharophimosis-ptosis-epicanthus inversus syndrome because of a chromosome 3q deletion.

Phelan–McDermid syndrome is a developmental disability syndrome with varying deletions of 22q13 and varying clinical severity. We tested the hypothesis that, in addition to loss of the telomeric gene SHANK3, specific genomic regions within 22q13 are associated with important clinical features. We used a customized oligo array comparative genomic hybridization of 22q12.3-terminus to obtain deletion breakpoints in a cohort of 70 patients with terminal 22q13 deletions. We used association and receiver operating characteristic statistical methods in a novel manner and also incorporated protein interaction networks to identify 22q13 genomic locations and genes associated with clinical features. Specific genomic regions and candidate genes within 22q13.2q13.32 were associated with severity of speech/language delay, neonatal hypotonia, delayed age at walking, hair-pulling behaviors, male genital anomalies, dysplastic toenails, large/fleshy hands, macrocephaly, short and tall stature, facial asymmetry, and atypical reflexes. We also found regions suggestive of a negative association with autism spectrum disorders. This work advances the field of research beyond the observation of a correlation between deletion size and phenotype and identifies candidate 22q13 loci, and in some cases specific genes, associated with singular clinical features observed in Phelan–McDermid syndrome. Our statistical approach may be useful in genotype–phenotype analyses for other microdeletion or microduplication syndromes. Genet Med 2014:16(4):318–328.

This study is the first to describe age-related changes in a large cohort of patients with Phelan-McDermid syndrome (PMS), also known as 22q13 deletion syndrome. Over a follow-up period of up to 12 years, physical examinations and structured interviews were conducted for 201 individuals diagnosed with PMS, 120 patients had a focused, high-resolution 22q12q13 array CGH, and 92 patients' deletions were assessed for parent-of-origin. 22q13 genomic anomalies include terminal deletions of 22q13 (89 %), terminal deletions and interstitial duplications (9 %), and interstitial deletions (2 %). Considering different age groups, in older patients, behavioral problems tended to subside, developmental abilities improved, and some features such as large or fleshy hands, full or puffy eyelids, hypotonia, lax ligaments, and hyperextensible joints were less frequent. However, the proportion reporting an autism spectrum disorder, seizures, and cellulitis, or presenting with lymphedema or abnormal reflexes increased with age. Some neurologic and dysmorphic features such as speech and developmental delay and macrocephaly correlated with deletion size. Deletion sizes in more recently diagnosed patients tend to be smaller than those diagnosed a decade earlier. Seventy-three percent of de novo deletions were of paternal origin. Seizures were reported three times more often among patients with a de novo deletion of the maternal rather than paternal chromosome 22. This analysis improves the understanding of the clinical presentation and natural history of PMS and can serve as a reference for the prevalence of clinical features in the syndrome.

Consequent to power sector reforms, customers' satisfaction is gaining vital importance at power distribution companies. Customers are now getting a choice of choosing their electricity supplier as well as options of investing in their own power generating equipment. The threat of losing customers is driving power distribution companies towards ensuring customer satisfaction. Apart from availability of electricity supply on a 24 / 7 basis, electricity customers now expect ease in getting new electricity connection, advice on most suitable category of supply, timely meter reading, billing, and handling of grievance. To provide customers with great satisfaction, power distribution companies have to give quality attention to offering excellent services that attracts customers and clear up all customers' complaints. Frontline managers play a very significant role in the electric utility companies. They act as an inter-face between the customers / public and the company. This chapter attempts to elaborate customers' expectations from the frontline managers in power distribution companies.

Autism spectrum disorders (ASDs) include three main conditions: autistic disorder (AD), pervasive developmental disorder, not otherwise specified (PDD-NOS), and Asperger syndrome. It has been shown that many genes associated with ASDs are involved in the neuroligin–neurexin interaction at the glutamate synapse: NLGN3, NLGN4, NRXN1, CNTNAP2, and SHANK3. We screened this last gene in two cohorts of ASD patients (133 patients from US and 88 from Italy). We found 5/221 (2.3%) cases with pathogenic alterations: a 106 kb deletion encompassing the SHANK3 gene, two frameshift mutations leading to premature stop codons, a missense mutation (p.Pro141Ala), and a splicing mutation (c.1820-4 G>A). Additionally, in 17 patients (7.7%) we detected a c.1304+48C>T transition affecting a methylated cytosine in a CpG island. This variant is reported as SNP rs76224556 and was found in both US and Italian controls, but it results significantly more frequent in our cases than in the control cohorts. The variant is also significantly more common among PDD-NOS cases than in AD cases. We also screened this gene in an independent replication cohort of 104 US patients with ASDs, in which we found a missense mutation (p.Ala1468Ser) in 1 patient (0.9%), and in 8 patients (7.7%) we detected the c.1304+48C>T transition. While SHANK3 variants are present in any ASD subtype, the SNP rs76224556 appears to be significantly associated with PDD-NOS cases. This represents the first evidence of a genotype–phenotype correlation in ASDs and highlights the importance of a detailed clinical-neuropsychiatric evaluation for the effective genetic screening of ASD patients.

Introduction Mesomelic dysplasias are a group of skeletal disorders characterised by shortness of the middle limb segments (mesomelia). They are divided into 11 different categories. Among those without known molecular basis is mesomelic dysplasia Savarirayan type, characterised by severe shortness of the middle segment of the lower limb. Objective To identify the molecular cause of mesomelic dysplasia Savarirayan type. Methods and results We performed array comparative genomic hybridisation in three unrelated patients with mesomelic dysplasia Savarirayan type and identified 2 Mb overlapping de novo microdeletions on chromosome 6p22.3. The deletions encompass four known genes: MBOAT1 , E2F3, CDKAL1 and SOX4. All patients showed mesomelia of the lower limbs with hypoplastic tibiae and fibulae. We identified a fourth patient with intellectual disability and an overlapping slightly larger do novo deletion also encompassing the flanking gene ID4. Given the fact that the fourth patient had no skeletal abnormalities and none of the genes in the deleted interval are known to be associated with abnormalities in skeletal development, other mutational mechanisms than loss of function of the deleted genes have to be considered. Analysis of the genomic region showed that the deletion removes two regulatory boundaries and brings several potential limb enhancers into close proximity of ID4. Thus, the deletion could result in the aberrant activation and misexpression of ID4 in the limb bud, thereby causing the mesomelic dysplasia. Conclusions Our data indicate that the distinct deletion 6p22.3 is associated with mesomelic dysplasia Savarirayan type featuring hypoplastic, triangular-shaped tibiae and abnormally shaped or hypoplastic fibulae.

CAR-T cell therapy has demonstrated remarkable success in long-term remission of relapsed or refractory B-ALL. Unlike western countries, most of the relapsed/refractory B-ALL patients in India are unable to afford allogeneic stem cell transplant (allo-SCT), and simply choose palliation. To assess the potential use of CAR-T cells as a first line therapy, it is critical to evaluate CAR-T cell efficacy in patients unable to undergo allo-SCT. As a first step, a novel anti-CD19 CAR was designed and CD19-vector was produced using lentiviral mediated gene delivery system. To generate CD19+CAR T cells, T cells from healthy subjects were transduced and expanded either with rIL-2 or rIL-15. Almost 20-50% transduced T cells express CD19-CAR on their surface as analysed by flow cytometry. Next, efficacy of CD19-CAR-T cells was examined by cytotoxicity assay using CD19+ malignant B cell lines (Raji cells) and autologous B cells. CAR-T cells were able to kill majority of CD19+B cells effectively (100% killing). In addition, CAR-T cells secreted high levels of IFNΓ and IL-2. The CAR-T cells expanded in presence of rIL-15 showed fewer Treg compared to CAR-T cells expanded using rIL-2, however, there were no differences in numbers of T stem cells (TSC) and central memory T cells (TCM). In addition to data on healthy volunteers reflected above, results of above tests in relapsed/ refractory B-ALL patients is ongoing and being collated for inclusion in the final analysis. In conclusion, our data demonstrate the successful development of an indigenous CAR-T cell platform for subsequent use in clinical trials of CD19+B cell malignancies. Citation Format: Alka Dwivedi, Sushant Kumar, Terry Fry, Gaurav Narula, Rahul Purwar. Evaluation of CD19 targeted T cells in relapsed or refractory ALL patients unable to afford allogenic bone marrow transplant in India [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 603. doi:10.1158/1538-7445.AM2017-603

A 10-year-old boy presented with a history of significant delay in language acquisition as well as receptive and expressive language impairment that persisted into elementary school. In school, he exhibited difficulty with reading comprehension, telling and understanding narratives, and making inferences. Other aspects of his neurodevelopment were normal, with no history of significant medical concerns. He did not have hearing impairment, oromotor dysfunction, or specific neurologic abnormalities. He did not meet testing criteria for autism. Chromosomal microarray analysis and quantitative polymerase chain reaction determined that he had a de novo 159-kilobase deletion of chromosome 16q24.1 that included the ATP2C2 gene. ATP2C2 is a known candidate gene for specific language impairment and is postulated to have neurobiological significance in memory-related circuits. Our patient's language deficits were consistent with a global type of specific language impairment impacting language comprehension, formulation, semantics, syntax, and phonology attributed to his de novo chromosome deletion.

Coffin-Lowry syndrome (CLS) is a rare X-linked dominant disorder characterized by intellectual disability, craniofacial abnormalities, short stature, tapering fingers, hypotonia, and skeletal malformations. CLS is caused by mutations in the Ribosomal Protein S6 Kinase, 90 kDa, Polypeptide 3 (RPS6KA3) gene located at Xp22.12, which encodes Ribosomal S6 Kinase 2 (RSK2). Here we analyzed RPS6KA3 in three unrelated CLS patients including one from the historical Coffin-Lowry syndrome family and found two novel mutations. To date, over 140 mutations in RPS6KA3 have been reported. However, the etiology of the very first familial case, which was described in 1971 by Lowry with detailed phenotype and coined the term CLS, has remained unknown. More than 40 years after the report, we succeeded in identifying deposited fibroblast cells from one patient of this historic family and found a novel heterozygous 216 bp in-frame deletion, encompassing exons 15 and 16 of RPS6KA3. Drop episodes in CLS patients were reported to be associated with truncating mutations deleting the C-terminal kinase domain (KD), and only one missense mutation and one single basepair duplication involving the C-terminal KD of RSK2 in the patients with drop episode have been reported thus far. Here we report the first in-frame deletion in C-terminal KD of RPS6KA3 in a CLS patient with drop episodes.

Previous studies have limited the use of specific X-chromosome array designed platforms to the evaluation of patients with intellectual disability. In this retrospective analysis, we reviewed the clinical utility of an X-chromosome array in a variety of scenarios. We divided patients according to the indication for the test into four defined categories: (1) autism spectrum disorders and/or developmental delay and/or intellectual disability (ASDs/DD/ID) with known family history of neurocognitive disorders; (2) ASDs/DD/ID without known family history of neurocognitive disorders; (3) breakpoint definition of an abnormality detected by a different cytogenetic test; and (4) evaluation of suspected or known X-linked conditions. A total of 59 studies were ordered with 27 copy number variants detected in 25 patients (25/59 = 42%). The findings were deemed pathogenic/likely pathogenic (16/59 = 27%), benign (4/59 = 7%) or uncertain (7/59 = 12%). We place particular emphasis on the utility of this test for the diagnostic evaluation of families affected with X-linked conditions and how it compares to whole genome arrays in this setting. In conclusion, the X-chromosome array frequently detects genomic alterations of the X chromosome and it has advantages when evaluating some specific X-linked conditions. However, careful interpretation and correlation with clinical findings is needed to determine the significance of such changes. When the X-chromosome array was used to confirm a suspected X-linked condition, it had a yield of 63% (12/19) and was useful in the evaluation and risk assessment of patients and families. © 2012 Wiley Periodicals, Inc.

Disruptions to LIS1 gene expression result in neuronal migration abnormalities. LIS1 heterozygosity is a significant cause of lissencephaly, while overexpression has recently been noted in cases of microcephaly, ventriculomegaly, and dysgenesis of the corpus callosum with normal cortical gyration. We report a partial LIS1 duplication in a child with microcephaly, neurodevelopmental delays, and profound white matter atrophy in the absence of overt lissencephaly. The duplicated genetic segment was contained entirely within the first intron of LIS1, a segment that often contains inducers of transcription. Normal gyral patterns with mild volume loss were observed at birth. Follow-up cranial imaging revealed further white matter loss, diminished sulcation, and ventriculomegaly, suggesting expanding hydrocephalus ex vacuo. The radiographic pattern has not been documented in the presence of a LIS1 gene abnormality, and suggests that altered expression of LIS1 has wider phenotypic manifestations than currently defined.

Split-hand/foot malformation with long-bone deficiency (SHFLD) is a relatively rare autosomal-dominant skeletal disorder, characterized by variable expressivity and incomplete penetrance. Although several chromosomal loci for SHFLD have been identified, the molecular basis and pathogenesis of most SHFLD cases are unknown. In this study we describe three unrelated kindreds, in which SHFLD segregated with distinct but overlapping duplications in 17p13.3, a region previously linked to SHFLD. In a large three-generation family, the disorder was found to segregate with a 254 kb microduplication; a second microduplication of 527 kb was identified in an affected female and her unaffected mother, and a 430 kb microduplication versus microtriplication was identified in three affected members of a multi-generational family. These findings, along with previously published data, suggest that one locus responsible for this form of SHFLD is located within a 173 kb overlapping critical region, and that the copy gains are incompletely penetrant.

Translocations involving the short arms of the X and Y chromosomes are rare and can result in a functional disomy of the short arm of the X chromosome, including the dosage-sensitive sex reversal (DSS) locus. A result of such imbalance may be sex reversal with multiple congenital anomalies. We present the clinical and cytogenetic evaluation of a newborn infant with DSS and additional clinical findings of minor facial anomalies, left abdominal mass, 5th finger clinodactyly, and mild hypotonia. The external genitalia appeared to be normal female. The infant had bilateral corneal opacities and findings suggestive of anterior segment dysgenesis. Ultrasonography showed a small uterus with undetectable ovaries, and a left multicystic dysplastic kidney. High-resolution chromosome analysis identified the presence of a derivative Y chromosome, 47,XY, +der(Y)t(X;Y)(p21.1;p11.2), which was confirmed by fluorescence in situ hybridization studies. Array CGH showed a 35.1 Mb copy number gain of chromosome region Xp22.33-p21.1 and a 52.2 Mb copy number gain of Yp11.2-qter, in addition to the intact X and Y chromosomes. Previously reported patients with XY sex reversal have not had DSS with corneal opacities, dysgenesis of the anterior segment of the eye, and unilateral multicystic dysplastic kidney. These findings represent a new form of XY sex reversal due to an Xp duplication.

Optima for growth, oxygenic photosynthesis and glutamine synthetase activity occurred at pH 10, thus suggesting that the cyanobacterium Hapalosiphon welwitschii is an alkalophile. It produced a Cu-Zn efflux system at pH 9 or 10, but not at pH 7 or 8, to relieve photosynthesis from Cu or Zn inhibition. This finding has a bearing on the ecophysiological competence of the cyanobacterium under natural conditions.

The phosphorus removal potential of the bloom‐forming, alkali‐tolerant cyanobacterium Oscillatoria amphibia was studied by entrapping and immobilizing it in calcium alginate. The uptake kinetics for orthophosphate‐phosphorus (PO4‐P) were determined by growing O. amphibia in Jaworski medium (pH 9) in 14.6 Wm‐2 light intensity at 24±1°C. The pH of the culture medium increased to 11.0 during growth. This facilitated the maintenance of unialgal cultures in the absence of any contaminants. Immobilized filaments showed potential capability of phosphorus accumulation. A stocking biomass of 0.4 μg chl a bead‐1 at external pH 9 was found to be suitable for rapid phosphorus removal. Phosphorus uptake kinetics of immobilized filaments were also dependent on the initial phosphorus concentration. These findings may be useful for designing a bioreactor to optimize phosphorus removal using an immobilized cyanobacterial system.

Lysophosphatidic acid (LPA), a potent bioactive phospholipid, is a natural component of food products like soy and egg yolk. LPA modulates a number of epithelial functions and has been shown to inhibit cholera toxin-induced diarrhea. Antidiarrheal effects of LPA are known to be mediated by inhibiting chloride secretion. However, the effects of LPA on chloride absorption in the mammalian intestine are not known. The present studies examined the effects of LPA on apical Cl−/OH− exchangers known to be involved in chloride absorption in intestinal epithelial cells. Caco-2 cells were treated with LPA, and Cl−/OH− exchange activity was measured as DIDS-sensitive 36Cl− uptake. Cell surface biotinylation studies were performed to evaluate the effect of LPA on cell surface levels of apical Cl−/OH− exchangers, downregulated in adenoma (DRA) (SLC26A3), and putative anion transporter-1 (SLC26A6). Treatment of Caco-2 cells with LPA (100 μM) significantly stimulated Cl−/OH− exchange activity. Specific agonist for LPA2 receptor mimicked the effects of LPA. LPA-mediated stimulation of Cl−/OH− exchange activity was dependent on activation of phosphatidylinositol 3-kinase/Akt signaling pathway. Consistent with the functional activity, LPA treatment resulted in increased levels of DRA on the apical membrane. Our results demonstrate that LPA stimulates apical Cl−/OH− exchange activity and surface levels of DRA in intestinal epithelial cells. This increase in Cl−/OH− exchange may contribute to the antidiarrheal effects of LPA.

Extra pulmonary tuberculosis (EPTB) constitutes around 20% of all tuberculosis cases in India. Conventional methods are of limited use in diagnosing this form of the disease. Polymerase chain reaction (PCR) has emerged as a sensitive and specific tool for documenting the presence of Mycobacterium tuberculosis in clinical samples but lacks quantitative ability. The present study evaluates peripheral blood as an alternative clinical specimen for diagnosing EPTB. Peripheral blood samples from 38 EPTB and 89 non tuberculous subjects were analyzed for the presence of tubercle bacilli by MPB 64 gene based PCR method. The assay gave an overall sensitivity of 60.53% with negative predictive value of 76.92% which is superior to present gold standard of mycobacterial culture (10.53 and 72.36%). Additionally, 43.82% of non tuberculous subjects gave positive results with the PCR, thus mitigating the clinical utility of this test. An in-house Competitive PCR (C-PCR) assay was used to determine the mycobacterial load in peripheral blood from culture positive, culture negative EPTB patients and non tuberculous controls which ranged from 7498-12498, 602-4797 and 101-800 genome equivalent (ge)/mL, respectively. The data clearly demonstrated that C-PCR assay can furnish insightful information in diagnosing extra pulmonary disease.

Ileal apical Na+-dependent bile acid transporter (ASBT) is responsible for reabsorbing the majority of bile acids from the intestinal lumen. Rapid adaptation of ASBT function in response to physiological and pathophysiological stimuli is essential for the maintenance of bile acid homeostasis. However, not much is known about molecular mechanisms responsible for acute posttranscriptional regulation of ileal ASBT. The protein kinase C (PKC)-dependent pathway represents a major cell signaling mechanism influencing intestinal epithelial functions. The present studies were, therefore, undertaken to investigate ASBT regulation in intestinal Caco-2 monolayers by the well-known PKC activator phorbol 12-myristate 13-acetate (PMA). Our results showed that Na+-dependent [3H]taurocholic acid uptake in Caco-2 cells was significantly inhibited in response to 2 h incubation with 100 nM PMA compared with incubation with 4α-PMA (inactive form). The inhibitory effect of PMA was blocked in the presence of 5 μM bisindolylmaleimide I (PKC inhibitor) but not 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid-AM (Ca2+chelator) or LY-294002 (phosphatidylinositol 3-kinase inhibitor). PMA inhibition of ASBT function was also abrogated in the presence of myristoylated PKCζ pseudosubstrate peptide, indicating involvement of the atypical PKCζ isoform. The inhibition by PMA was associated with a significant decrease in the maximal velocity of the transporter and a reduction in ASBT plasma membrane content, suggesting a modulation by vesicular recycling. Our novel findings demonstrate a posttranscriptional modulation of ileal ASBT function and membrane expression by phorbol ester via a PKCζ-dependent pathway.

Monocarboxylate transporter (MCT1) plays an important role in the absorption of short-chain fatty acids (SCFA) such as butyrate in the human colon. Previous studies from our laboratory have demonstrated that phorbol ester, PMA (1 μM, 24 h), upregulates butyrate transport and MCT1 protein expression in human intestinal Caco-2 cells. However, the molecular mechanisms involved in the transcriptional regulation of MCT1 gene expression by PMA in the intestine are not known. In the present study, we showed that PMA (0.1 μM, 24 h) increased the MCT1 promoter activity (−871/+91) by approximately fourfold. A corresponding increase in MCT1 mRNA abundance in response to PMA was also observed. PMA-induced stimulation of MCT1 promoter activity was observed as early as 1 h and persisted until 24 h, suggesting that the effects of PMA are attributable to initial PKC activation. Kinase inhibitor and phosphorylation studies indicated that these effects may be mediated through activation of the atypical PKC-ζ isoform. 5′-deletion studies demonstrated that the MCT1 core promoter region (−229/+91) is the PMA-responsive region. Site-directed mutagenesis studies showed the predominant involvement of potential activator protein 2 (AP2) binding site in the activation of MCT1 promoter activity by PMA. In addition, overexpression of AP2 in Caco-2 cells significantly increased MCT1 promoter activity in a dose-dependent manner. These findings showing the regulation of MCT1 promoter by PKC and AP2 are of significant importance for an understanding of the molecular regulation of SCFA absorption in the human intestine.

Serotonin or 5-hydroxytryptamine (5-HT) influences numerous functions in the gastrointestinal tract. We previously demonstrated that 5-HT treatment of Caco-2 cells inhibited Na+/H+exchangers (NHE) and Cl−/OH−exchange activities via distinct signaling mechanisms. Since regulation of several ion transporters such as NHE3 is influenced by intact cytoskeleton, we hypothesized that 5-HT modifies actin cytoskeleton and/or brush-border membrane architecture via involvement of signaling pathways. Ultrastructural analysis showed that 5-HT (0.1 μM, 1 h) treatment of Caco-2 cells caused the apical membrane to assume a convex dome shape that was associated with shortening of microvilli. To examine whether these cellular architecture changes are cytoskeleton driven, we analyzed actin cytoskeleton by fluorescence microscopy. 5-HT induced basal stress fibers with prominent cortical actin filaments via 5-HT3 and 5-HT4 receptor subtypes. This induction was partially attenuated by chelation of intracellular Ca2+and PKCα inhibition (Go6976). In vitro assays revealed that PKCα interacted with actin and this association was increased by 5-HT. Our data provide novel evidence that 5-HT-induced signaling via 5-HT3/4 receptor subtypes to cause Ca2+and PKCα-dependent regulation of actin cytoskeleton may play an important role in modulation of ion transporters that contribute to pathophysiology of diarrheal conditions associated with elevated levels of 5-HT.

Niemann-Pick C1-like 1 (NPC1L1) is an essential intestinal component of cholesterol absorption. However, little is known about the molecular regulation of intestinal NPC1L1 expression and promoter activity. We demonstrated that human NPC1L1 mRNA expression was significantly decreased by 25-hydroxycholesterol but increased in response to cellular cholesterol depletion achieved by incubation with Mevinolin (an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase) in human intestinal Caco-2 cells. We also showed that a −1741/+56 fragment of the NPC1L1 gene demonstrated high promoter activity in Caco-2 cells that was reduced by 25-hydroxycholesterol and stimulated by cholesterol depletion. Interestingly, we showed that the NPC1L1 promoter is remarkably transactivated by the overexpression of sterol regulatory element (SRE) binding protein (SREBP)-2, suggesting its involvement in the sterol-induced alteration in NPC1L1 promoter activity. Finally, we identified two putative SREs in the human NPC1L1 promoter and established their essential roles in mediating the effects of cholesterol on promoter activity. Our study demonstrated the modulation of human NPC1L1 expression and promoter activity by cholesterol in a SREBP-2-dependent mechanism.