The probable reasons of arsenic susceptibility in a chronically exposed population of West Bengal.

Arsenic is potent human carcinogen which affects millions of people across the globe. Arsenic induced pre-cancerous and cancerous skin lesions are hall marks of chronic arsenic toxicity. Even then, only 15%-20% of the population manifest arsenic-induced skin lesions but the rest do not, the reason for which in not very clear. Not only that, conjunctival irritations of the eyes, peripheral neuropathy and respiratory distress are the non-dermatological health effects which are often manifested in them in addition to the cancers of skin and other internal organs. In this work we have considered 233 arsenic exposed individuals with skin lesions and 205 arsenic exposed individuals without skin lesions from the highly arsenic affected Murshidabad district of West Bengal. We have compared arsenic exposure in the two groups through drinking water. Both the study groups have similar levels of arsenic exposure, drinking same arsenic laden water. Results show that higher amounts of arsenic were retained in the nails and hair of the skin lesion group compared to the no skin lesion group. Significant higher amounts of chromosomal aberration and micronucleus formation were found in the skin lesion group, than the no skin lesion group. Incidences of conjunctival irritations of the eyes, peripheral neuropathy and respiratory distress were much higher in the former group compared to the later. We, thus found that one group was more susceptible than the other, even with similar levels of arsenic exposure. We have tried to identify and discuss the probable reasons for this observation with reference to our previous works in the exposed population from West Bengal, India.

Pharmacologic and Genetic Downregulation of Proprotein Convertase Subtilisin/Kexin Type 9 and Survival From Sepsis.

OBJECTIVES: Treatments that prevent sepsis complications are needed. Circulating lipid and protein assemblies-lipoproteins play critical roles in clearing pathogens from the bloodstream. We investigated whether early inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) may accelerate bloodstream clearance of immunogenic bacterial lipids and improve sepsis outcomes. DESIGN: Genetic and clinical epidemiology, and experimental models. SETTING: Human genetics cohorts, secondary analysis of a phase 3 randomized clinical trial enrolling patients with cardiovascular disease (Evaluation of Cardiovascular Outcomes After an Acute Coronary Syndrome During Treatment With Alirocumab [ODYSSEY OUTCOMES]; NCT01663402), and experimental murine models of sepsis. PATIENTS OR SUBJECTS: Nine human cohorts with sepsis (total n = 12,514) were assessed for an association between sepsis mortality and PCSK9 loss-of-function (LOF) variants. Incident or fatal sepsis rates were evaluated among 18,884 participants in a post hoc analysis of ODYSSEY OUTCOMES. C57BI/6J mice were used in Pseudomonas aeruginosa and Staphylococcus aureus bacteremia sepsis models, and in lipopolysaccharide-induced animal models. INTERVENTIONS: Observational human cohort studies used genetic PCSK9 LOF variants as instrumental variables. ODYSSEY OUTCOMES participants were randomized to alirocumab or placebo. Mice were administered alirocumab, a PCSK9 inhibitor, at 5 mg/kg or 25 mg/kg subcutaneously, or isotype-matched control, 48 hours prior to the induction of bacterial sepsis. Mice did not receive other treatments for sepsis. MEASUREMENTS AND MAIN RESULTS: Across human cohort studies, the effect estimate for 28-day mortality after sepsis diagnosis associated with genetic PCSK9 LOF was odds ratio = 0.86 (95% CI, 0.67-1.10; p = 0.24). A significant association was present in antibiotic-treated patients. In ODYSSEY OUTCOMES, sepsis frequency and mortality were infrequent and did not significantly differ by group, although both were numerically lower with alirocumab vs. placebo (relative risk of death from sepsis for alirocumab vs. placebo, 0.62; 95% CI, 0.32-1.20; p = 0.15). Mice treated with alirocumab had lower endotoxin levels and improved survival. CONCLUSIONS: PCSK9 inhibition may improve clinical outcomes in sepsis in preventive, pretreatment settings.

Converging evidence from exome sequencing and common variants implicates target genes for osteoporosis.

In this study, we leveraged the combined evidence of rare coding variants and common alleles to identify therapeutic targets for osteoporosis. We undertook a large-scale multiancestry exome-wide association study for estimated bone mineral density, which showed that the burden of rare coding alleles in 19 genes was associated with estimated bone mineral density (P < 3.6 × 10-7). These genes were highly enriched for a set of known causal genes for osteoporosis (65-fold; P = 2.5 × 10-5). Exome-wide significant genes had 96-fold increased odds of being the top ranked effector gene at a given GWAS locus (P = 1.8 × 10-10). By integrating proteomics Mendelian randomization evidence, we prioritized CD109 (cluster of differentiation 109) as a gene for which heterozygous loss of function is associated with higher bone density. CRISPR-Cas9 editing of CD109 in SaOS-2 osteoblast-like cell lines showed that partial CD109 knockdown led to increased mineralization. This study demonstrates that the convergence of common and rare variants, proteomics and CRISPR can highlight new bone biology to guide therapeutic development.

Gamma-Aminobutyric Acid Type A Receptor Variants are Associated with Autism Spectrum Disorders.

Despite several efforts to identify the causes of autism spectrum disorders (ASD), its etiology remains still unclear. Among other aspects, genes that encode neurotransmitter receptors are strong candidates for autism. Here, we wanted to study some genetic variants of gamma-aminobutyric acid (GABA) receptor subunit genes GABRB3, GABRG3, and GABRA5, located on chromosome 15q11-q13 that might contribute to the etiology of ASD in the affected children of West Bengal. rs7180158, rs2081648 (GABRB3); rs12910555 (GABRG3); rs35399885, rs35832850 (GABRA5) were analyzed in 316 children with ASD and 227 healthy controls. Phenotypic associations were evaluated by Childhood Autism Rating Scale (CARS). Gene expression levels were measured by quantitative real-time PCR. ASD probands showed a higher frequency of "A" allele for rs7180158, "G" allele for rs12901555, and "T" allele for rs35399885. The GA + AA genotypes (rs7180158) and CT + TT genotypes (rs35399885) were found to confer significant risk towards ASD. rs2081648 was found to have transmission bias in the family. Additionally, these variants were found to be associated with one or more of ASD-associated phenotypic traits. Multifactor dimensionality reduction (MDR) analyses showed mostly independent contributory effects of some of the variants. Again, the gene expression levels of GABRB3, GABRG3, and GABRA5 were downregulated in the cases than the controls. ForGABRA5 rs35399885, the CC genotypes corresponded to higher expression levels compared to the other groups. This study reveals that genetic variants of GABAA receptor subunit genes are significantly associated with ASD. No data for the mentioned variants are found in the population of West Bengal, India.

Liquid antisolvent crystallization of pharmaceutical compounds: current status and future perspectives.

The present work reviews the liquid antisolvent crystallization (LASC) to prepare the nanoparticle of pharmaceutical compounds to enhance their solubility, dissolution rate, and bioavailability. The application of ultrasound and additives is discussed to prepare the particles with narrow size distribution. The use of ionic liquid as an alternative to conventional organic solvent is presented. Herbal compounds, also known for low aqueous solubility and limited clinical application, have been crystalized by LASC and discussed here. The particle characteristics such as particle size and particle size distribution are interpreted in terms of supersaturation, nucleation, and growth phenomena. To overcome the disadvantage of batch crystallization, the scientific literature on continuous flow reactors is also reviewed. LASC in a microfluidic device is emerging as a promising technique. The different design of the microfluidic device and their application in LASC are discussed. The combination of the LASC technique with traditional techniques such as high-pressure homogenization and spray drying is presented. A comparison of product characteristics prepared by LASC and the supercritical CO2 antisolvent method is discussed to show that LASC is an attractive and inexpensive alternative for nanoparticle preparation. One of the major strengths of this paper is a discussion on less-explored applications of LASC in pharmaceutical research to attract the attention of future researchers.

Common and rare variant associations with clonal haematopoiesis phenotypes.

Clonal haematopoiesis involves the expansion of certain blood cell lineages and has been associated with ageing and adverse health outcomes1-5. Here we use exome sequence data on 628,388 individuals to identify 40,208 carriers of clonal haematopoiesis of indeterminate potential (CHIP). Using genome-wide and exome-wide association analyses, we identify 24 loci (21 of which are novel) where germline genetic variation influences predisposition to CHIP, including missense variants in the lymphocytic antigen coding gene LY75, which are associated with reduced incidence of CHIP. We also identify novel rare variant associations with clonal haematopoiesis and telomere length. Analysis of 5,041 health traits from the UK Biobank (UKB) found relationships between CHIP and severe COVID-19 outcomes, cardiovascular disease, haematologic traits, malignancy, smoking, obesity, infection and all-cause mortality. Longitudinal and Mendelian randomization analyses revealed that CHIP is associated with solid cancers, including non-melanoma skin cancer and lung cancer, and that CHIP linked to DNMT3A is associated with the subsequent development of myeloid but not lymphoid leukaemias. Additionally, contrary to previous findings from the initial 50,000 UKB exomes6, our results in the full sample do not support a role for IL-6 inhibition in reducing the risk of cardiovascular disease among CHIP carriers. Our findings demonstrate that CHIP represents a complex set of heterogeneous phenotypes with shared and unique germline genetic causes and varied clinical implications.

Birth related parameters are important contributors in autism spectrum disorders.

Autism spectrum disorders is a group of childhood onset neurodevelopmental disorders affecting millions of children across the globe. Characterised by age inappropriate lack of reciprocal social interaction, repetitive behaviours and deficits in communication skills, it has been found to have genetic, epigenetic and environmental contributions. In this work, we wanted to identify the effects of birth related parameters on the disease pathogenesis in an exposed population of West Bengal, India. We have considered age of both parents at birth, difference in parental age, familial history of mental illness, delay in developmental-milestones, birth-weight, birth-order, birth-term, mode of delivery and gestational complications as contributors. We found the parental age and their age difference to be the most important contributors towards ASD in this population. Birth order, sex of the probands, complications during gestation, birth weight, family history of mental illness and birth history also contributed to the condition, although to a lesser extent. Since such types of data are lacking in Indian population, this report adds useful information to the relevant field.

Genome-wide analysis provides genetic evidence that ACE2 influences COVID-19 risk and yields risk scores associated with severe disease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters human host cells via angiotensin-converting enzyme 2 (ACE2) and causes coronavirus disease 2019 (COVID-19). Here, through a genome-wide association study, we identify a variant (rs190509934, minor allele frequency 0.2-2%) that downregulates ACE2 expression by 37% (P = 2.7 × 10-8) and reduces the risk of SARS-CoV-2 infection by 40% (odds ratio = 0.60, P = 4.5 × 10-13), providing human genetic evidence that ACE2 expression levels influence COVID-19 risk. We also replicate the associations of six previously reported risk variants, of which four were further associated with worse outcomes in individuals infected with the virus (in/near LZTFL1, MHC, DPP9 and IFNAR2). Lastly, we show that common variants define a risk score that is strongly associated with severe disease among cases and modestly improves the prediction of disease severity relative to demographic and clinical factors alone.

Exome sequencing and analysis of 454,787 UK Biobank participants.

A major goal in human genetics is to use natural variation to understand the phenotypic consequences of altering each protein-coding gene in the genome. Here we used exome sequencing1 to explore protein-altering variants and their consequences in 454,787 participants in the UK Biobank study2. We identified 12 million coding variants, including around 1 million loss-of-function and around 1.8 million deleterious missense variants. When these were tested for association with 3,994 health-related traits, we found 564 genes with trait associations at P ≤ 2.18 × 10-11. Rare variant associations were enriched in loci from genome-wide association studies (GWAS), but most (91%) were independent of common variant signals. We discovered several risk-increasing associations with traits related to liver disease, eye disease and cancer, among others, as well as risk-lowering associations for hypertension (SLC9A3R2), diabetes (MAP3K15, FAM234A) and asthma (SLC27A3). Six genes were associated with brain imaging phenotypes, including two involved in neural development (GBE1, PLD1). Of the signals available and powered for replication in an independent cohort, 81% were confirmed; furthermore, association signals were generally consistent across individuals of European, Asian and African ancestry. We illustrate the ability of exome sequencing to identify gene-trait associations, elucidate gene function and pinpoint effector genes that underlie GWAS signals at scale.

A critical review on the particle generation and other applications of rapid expansion of supercritical solution.

The novel particle generation processes of Active Pharmaceutical Ingredient (API)/drug have been extensively explored in recent decades due to their wide-range applications in the pharmaceutical industry. The Rapid Expansion of Supercritical Solutions (RESS) is one of the promising techniques to obtain the fine particles (micro to nano-size) of APIs with narrow particle size distribution (PSD). In RESS, supercritical carbon dioxide (SC CO2) and API are used as solvent and solute respectively. In this literature survey, the application of RESS in the formation of fine particles is critically reviewed. Solubility of API in SC CO2 and supersaturation are the key factors in tuning the particle size. The different approaches to model and predict the solubility of API in SC CO2 are discussed. Then, the effect of process parameters on mean particle size and the particle size distribution are interpreted in the context of solubility and supersaturation. Furthermore, the less-explored applications of RESS in preparation of solid-lipid nanoparticles, liposome, polymorphic conversion, cocrystallization and inclusion complexation are compared with traditional processes. The solubility enhancement of API in SC CO2 using co-solvent and its applications in particle generation are explored in published literature. The development and modifications in the conventional RESS process to overcome the limitations of RESS are presented. Finally, the perspective on RESS with special attention to its commercial operation is highlighted.

Pan-ancestry exome-wide association analyses of COVID-19 outcomes in 586,157 individuals.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), a respiratory illness that can result in hospitalization or death. We used exome sequence data to investigate associations between rare genetic variants and seven COVID-19 outcomes in 586,157 individuals, including 20,952 with COVID-19. After accounting for multiple testing, we did not identify any clear associations with rare variants either exome wide or when specifically focusing on (1) 13 interferon pathway genes in which rare deleterious variants have been reported in individuals with severe COVID-19, (2) 281 genes located in susceptibility loci identified by the COVID-19 Host Genetics Initiative, or (3) 32 additional genes of immunologic relevance and/or therapeutic potential. Our analyses indicate there are no significant associations with rare protein-coding variants with detectable effect sizes at our current sample sizes. Analyses will be updated as additional data become available, and results are publicly available through the Regeneron Genetics Center COVID-19 Results Browser.

An Association Study of Gamma-Aminobutyric Acid Type A Receptor Variants and Susceptibility to Autism Spectrum Disorders.

In this pilot study, we aim to identify the role of few genetic variants of GABA-receptor type A subunits GABRB3 (rs4906902, rs7171660), GABRG3 (rs208129, rs140679), GABRA5 (rs 140681) in the aetiology of autism spectrum disorders in a population of West Bengal. 192 ASD probands, their parents and 184 ethnically-matched healthy controls were recruited for the study. The rs4906902G and the rs140679T conferred significant risk towards ASD. rs7171660 and rs140679 had transmission bias in the family. Neither alleles of rs 208129 and rs 140681 showed significant over-representation in either groups. All these variants were associated with at least one deficit in ASD-associated phenotypes like 'relating to people', 'Imitation', 'emotional response', 'body use', 'taste, smell, touch response' and 'activity levels'.

Exome sequencing and characterization of 49,960 individuals in the UK Biobank.

The UK Biobank is a prospective study of 502,543 individuals, combining extensive phenotypic and genotypic data with streamlined access for researchers around the world1. Here we describe the release of exome-sequence data for the first 49,960 study participants, revealing approximately 4 million coding variants (of which around 98.6% have a frequency of less than 1%). The data include 198,269 autosomal predicted loss-of-function (LOF) variants, a more than 14-fold increase compared to the imputed sequence. Nearly all genes (more than 97%) had at least one carrier with a LOF variant, and most genes (more than 69%) had at least ten carriers with a LOF variant. We illustrate the power of characterizing LOF variants in this population through association analyses across 1,730 phenotypes. In addition to replicating established associations, we found novel LOF variants with large effects on disease traits, including PIEZO1 on varicose veins, COL6A1 on corneal resistance, MEPE on bone density, and IQGAP2 and GMPR on blood cell traits. We further demonstrate the value of exome sequencing by surveying the prevalence of pathogenic variants of clinical importance, and show that 2% of this population has a medically actionable variant. Furthermore, we characterize the penetrance of cancer in carriers of pathogenic BRCA1 and BRCA2 variants. Exome sequences from the first 49,960 participants highlight the promise of genome sequencing in large population-based studies and are now accessible to the scientific community.

MicroRNAs play an important role in contributing to arsenic susceptibility in the chronically exposed individuals of West Bengal, India.

Arsenic exposure by groundwater contamination is a menace which threatens more than 26 million individuals of West Bengal. Interestingly, with similar levels of arsenic exposure, only 15-20% of the population show arsenic-induced skin lesions, the hallmarks of chronic arsenic toxicity, but the rest do not. In this study, our aim was to identify whether microRNAs (miRNA) have any role to play in causing such arsenic susceptibility. Global plasma miRNA profiling was done in 12 arsenic-exposed individuals with skin lesions and 12 exposed individuals without skin lesions. Two hundred two miRNAs were found to be differentially regulated between the two study groups. Results were validated by quantitative real-time PCR in 30 exposed subjects from each of the groups, which showed that among others miR-21, miR-23a, miR-27a, miR-122, miR-124, miR-126, miR-619, and miR-3613 were significantly upregulated and miR-1282 and miR-4530 were downregulated in the skin lesion group compared with the no skin lesion group. Bioinformatic analyses predicted that these altered miRNAs have targets in 7 different biochemical pathways, including glycerophospholipid metabolism, colorectal cancer, glycosphingolipid biosynthesis, T cell receptor signaling, and neurotrophin signaling pathways; glycerophospholipid metabolism pathway being the most enriched pathway. Association study show that these microRNAs contribute significantly to the increased prevalence of other non-dermatological health effects like conjunctival irritations of the eyes and respiratory distress in the study subjects. To our knowledge, this is the first study of its kind involving miRNA expressions contributing to arsenic susceptibility in the exposed population of West Bengal.

Evaluation of health effects, genetic damage and telomere length in children exposed to arsenic in West Bengal, India.

Increasing evidence of arsenic contamination in ground water and its associated adverse health outcomes affects millions of people worldwide. However, arsenic toxicity studies in children have gained impetus very recently due to the non-prominence of the hallmarks of arsenic toxicity i.e skin lesions. We recognized the need to evaluate the status of genetic damage brought about by early life exposure to arsenic in children as measured by micronucleus (MN) assay for three cell types namely buccal mucosa, urothelial cells and lymphocytes. A thorough health checkup and complete haematogram of the study participants was performed to measure overall health effects and changes in the blood profile in children exposed to arsenic through drinking water in West Bengal, India. Since telomere length alteration has been identified as a good indicator of arsenic toxicity in adults, we measured the telomere length of the arsenic exposed and unexposed children. We found that all the three cell types had significantly higher (P < 0.0001) MN frequency in the arsenic exposed children when compared to the unexposed. Blood profiling showed significantly altered neutrophil, eosinophil, lymphocyte and haemoglobin levels in the arsenic exposed children than their unexposed counterparts. Telomere length in the arsenic exposed children was slightly higher than the unexposed. This is a firsthand report of the genetic damage observed in children exposed to arsenic through drinking water in West Bengal, India.

Phytochemical investigations and evaluation of antimutagenic activity of the alcoholic extract of Glycosmis pentaphylla and Tabernaemontana coronaria by Ames test.

Chemical investigation of root bark of Glycosmis pentaphylla and stem bark of Tabernaemontana coronaria led to the isolation of three carbazole alkaloids glycozoline, glycozolidine and methyl carbazole 3-carboxylate, two furoquinoline alkaloids skimmianine and dictamine, an acridone alkaloid arborinine, three monomeric indole alkaloids coronaridine, 10-methoxy coronaridine and tabernaemontanine, and two dimeric indole alkaloids voacamine and tabernaelegantine B. Their structures were established by detailed spectral analysis. Mutagenic and antimutagenic potential of methanol extract of both plant materials were evaluated by Ames test against known positive mutagens 2-aminofluorine, 4-nitro-O-phenylenediamine and sodium azide using Salmonella typhimurium TA 98 and TA 100 bacterial strains both in the presence and absence of S9. Both the extracts were non-mutagenic in nature. Both the extracts of G. pentaphylla and T. coronaria exhibited significant antimutagenic activity against NPD and sodium azide for S. typhimurium TA98 and TA100 strains. The results indicated that the extracts could counteract the mutagenicity induced by different genotoxic compounds.

EPHX1 Y113H polymorphism is associated with increased risk of chronic obstructive pulmonary disease in Kazakhstan population.

Chronic obstructive pulmonary disease (COPD) is a type of obstructive lung disease characterized by long term poor airflow which worsens over time. It is considered to be one of the top five chronic diseases of the world in terms of morbidity and mortality. Genetic variability has been found to contribute to the development of COPD. Although association between gene polymorphisms in EPHX1 and TNF-a genes and chronic obstructive pulmonary disease (COPD) have been found but till date no genetic association studies have been done in the COPD affected Kazakhstan population. The aim of the present work was to investigate the association between the Y113H polymorphism (rs1051740) in EPHX1 gene and -308G/A polymorphism (rs1800629) in TNF-a gene and COPD in Kazakhstan population. A case-control study was conducted in Astana and Akmola regions of Kazakhstan, involving 55 cases with COPD and 52 healthy individuals who served as the controls. The polymorphisms were determined using conventional PCR and Sanger sequencing method. Results show that for the EPHX1 gene Y113H polymorphism, the presence of an "C" allele (TC/CC genotype) was significantly overrepresented in the COPD patients compared to the controls. For the TNF-a gene -308G/A polymorphism, no significant difference was found between the two groups. Thus we found that, Y113H polymorphism in EPHX1 gene contributed to increased susceptibility to COPD in the Kazakhstan population.

Increased microRNA 21 expression contributes to arsenic induced skin lesions, skin cancers and respiratory distress in chronically exposed individuals.

More than 26 million people in West Bengal, India, are exposed to arsenic through drinking water, leading to several deleterious endpoints including precancerous and cancerous skin lesions and other non-dermatological health effects. Here, our aim was to identify whether miR21 is associated with such dermatological and non-dermatological health outcomes in chronically exposed humans. A total of 123 subjects from West Bengal were recruited for this study (45 exposed individuals with skin lesions, 38 exposed individuals without skin lesions and 40 unexposed individuals). The miR21 expression patterns in the lymphocytes were studied by quantitative realtime PCR and the effects on downstream targets were validated by Western blotting. Associations between the miR21 expression patterns and non-dermatological health effects were determined from epidemiological survey data. In vitro studies were done with low dose (0.05ppm) of chronic arsenic exposure to HaCaT cells for 15 passages. Interestingly, within the exposed group, the skin lesion individuals showed almost 4.5 fold up-regulation of miR21 compared to the no skin lesion group. The expression of the downstream targets of miR21 (PTEN and PDCD4) varied inversely, while the expression of pAKT and PI3K varied proportionately with its expression levels. Results of in vitro studies showed similar trends. Again miR21 was 2.03 fold up-regulated in the exposed individuals with respiratory diseases compared to the individuals without the same. This study for the first time shows that miR21 plays an important role in contributing to arsenic induced dermatological and non-dermatological health outcomes in an exposed population.

Brief-exposure to preoperative bevacizumab reveals a TGF-ß signature predictive of response in HER2-negative breast cancers.

To best define biomarkers of response, and to shed insight on mechanism of action of certain clinically important agents for early breast cancer, we used a brief-exposure paradigm in the preoperative setting to study transcriptional changes in patient tumors that occur with one dose of therapy prior to combination chemotherapy. Tumor biopsies from breast cancer patients enrolled in two preoperative clinical trials were obtained at baseline and after one dose of bevacizumab (HER2-negative), trastuzumab (HER2-positive) or nab-paclitaxel, followed by treatment with combination chemo-biologic therapy. RNA-Sequencing based PAM50 subtyping at baseline of 46 HER2-negative patients revealed a strong association between the basal-like subtype and pathologic complete response (pCR) to chemotherapy plus bevacizumab (p ≤ 0.0027), but did not provide sufficient specificity to predict response. However, a single dose of bevacizumab resulted in down-regulation of a well-characterized TGF-ß activity signature in every single breast tumor that achieved pCR (p ≤ 0.004). The TGF-ß signature was confirmed to be a tumor-specific read-out of the canonical TGF-ß pathway using pSMAD2 (p ≤ 0.04), with predictive power unique to brief-exposure to bevacizumab (p ≤ 0.016), but not trastuzumab or nab-paclitaxel. Down-regulation of TGF-ß activity was associated with reduction in tumor hypoxia by transcription and protein levels, suggesting therapy-induced disruption of an autocrine-loop between tumor stroma and malignant cells. Modulation of the TGF-ß pathway upon brief-exposure to bevacizumab may provide an early functional readout of pCR to preoperative anti-angiogenic therapy in HER2-negative breast cancer, thus providing additional avenues for exploration in both preclinical and clinical settings with these agents.