Legionella pneumophila usurps host cell lipids for vacuole expansion and bacterial growth.

Vacuolar pathogens reside in membrane-bound compartments within host cells. Maintaining the integrity of this compartment is paramount to bacterial survival and replication as it protects against certain host surveillance mechanisms that function to eradicate invading pathogens. Preserving this compartment during bacterial replication requires expansion of the vacuole membrane to accommodate the increasing number of bacteria, and yet, how this is accomplished remains largely unknown. Here, we show that the vacuolar pathogen Legionella pneumophila exploits multiple sources of host cell fatty acids, including inducing host cell fatty acid scavenging pathways, in order to promote expansion of the replication vacuole and bacteria growth. Conversely, when exogenous lipids are limited, the decrease in host lipid availability restricts expansion of the replication vacuole membrane, resulting in a higher density of bacteria within the vacuole. Modifying the architecture of the vacuole prioritizes bacterial growth by allowing the greatest number of bacteria to remain protected by the vacuole membrane despite limited resources for its expansion. However, this trade-off is not without risk, as it can lead to vacuole destabilization, which is detrimental to the pathogen. However, when host lipid resources become extremely scarce, for example by inhibiting host lipid scavenging, de novo biosynthetic pathways, and/or diverting host fatty acids to storage compartments, bacterial replication becomes severely impaired, indicating that host cell fatty acid availability also directly regulates L. pneumophila growth. Collectively, these data demonstrate dual roles for host cell fatty acids in replication vacuole expansion and bacterial proliferation, revealing the central functions for these molecules and their metabolic pathways in L. pneumophila pathogenesis.

Overexpression of ARM repeat/U-box containing E3 ligase, PUB2 positively regulates growth and oxidative stress response in Arabidopsis.

Plant growth and development are governed by selective protein synthesis and degradation. Ubiquitination mediated protein degradation is governed by activating enzyme E1 followed by conjugating enzyme E2 and E3 ligase. Plant Armadillo (ARM) repeat/U-box (PUB) protein family is one of the important classes of E3 ligase. We studied the function of AtPUB2 by loss-of-function (knockout and knock down mutants) and gain-of-function (CaMV 35S promoter driven overexpression lines) approach in Arabidopsis. Under normal growth condition, we observed that loss-of-function mutant plants did not show any significant difference in growth when compared with wild-type possibly due to functional redundancy between PUB2 and PUB4. However, AtPUB2-OE lines exhibit early flowering and improved vegetative growth. Also, AtPUB2-OE seedlings showed sensitive phenotype in the presence of exogenous cytokinin. We found that AtPUB2 expression is induced under oxidative stress. Subcellular localization analysis shows that AtPUB2 is predominantly localized in the nucleus. We performed the phenotypic analysis under oxidative stress condition induced by methyl viologen (MV) and observed that overexpression lines display tolerance to oxidative stress in light and dark conditions. Furthermore, we found less amount of ROS accumulation, enhanced proline accumulation and decreased levels of MDA after MV treatment in AtPUB2-OE lines. PUB2-OE lines showed enhanced oxidative stress marker genes expression. By in vitro auto-ubiquitination assay, we also show that it possesses the E3 ligase activity. Overall, our findings suggest the possible role of AtPUB2 in plants ability to tolerate oxidative stress by enhancing the activity of antioxidant enzymes, which in turn improves ROS scavenging activity and homeostasis.

Calcium imaging: a technique to monitor calcium dynamics in biological systems.

Calcium ion (Ca2+) is a multifaceted signaling molecule that acts as an important second messenger. During the course of evolution, plants and animals have developed Ca2+ signaling in order to respond against diverse stimuli, to regulate a large number of physiological and developmental pathways. Our understanding of Ca2+ signaling and its components in physiological phenomena ranging from lower to higher organisms, and from single cell to multiple tissues has grown exponentially. The generation of Ca2+ transients or signatures for various stress factor is a well-known mechanism adopted in plant and animal systems. However, the decoding of such remarkable signatures is an uphill task and is always an interesting goal for the scientific community. In the past few decades, studies on the concentration and dynamics of intracellular Ca2+ are significantly increasing and have become a trend in modern biology. The advancement in approaches from Ca2+ binding dyes to in vivo Ca2+ imaging through the use of Ca2+ biosensors to achieve spatio-temporal resolution in micro and milliseconds range, provide us phenomenal opportunities to study live cell Ca2+ imaging or dynamics. Here, we describe the usage, improvement and advancement of Ca2+ based dyes, genetically encoded probes and sensors to achieve extraordinary Ca2+ imaging in plants and animals.

Scalable Asymmetric Syntheses of Foslevodopa and Foscarbidopa Drug Substances for the Treatment of Parkinson's Disease.

Foslevodopa (FLD, levodopa 4'-monophosphate, 3) and foscarbidopa (FCD, carbidopa 4'-monophosphate, 4) were identified as water-soluble prodrugs of levodopa (LD, 1) and carbidopa (CD, 2), respectively, which are useful for the treatment of Parkinson's disease. Herein, we describe asymmetric syntheses of FLD (3) and FCD (4) drug substances and their manufacture at pilot scale. The synthesis of FLD (3) employs a Horner-Wadsworth-Emmons olefination reaction followed by enantioselective hydrogenation of the double bond as key steps to introduce the α-amino acid moiety with the desired stereochemistry. The synthesis of FCD (4) features a Mizoroki-Heck reaction followed by enantioselective hydrazination to install the quaternary chiral center bearing a hydrazine moiety.

Novel Systemic Treatment Modalities Including Immunotherapy and Molecular Targeted Therapy for Recurrent and Metastatic Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinomas (HNSCCs) are the sixth most common cancers worldwide. More than half of patients with HNSCC eventually experience disease recurrence and/or metastasis, which can threaten their long-term survival. HNSCCs located in the oral cavity and larynx are usually associated with tobacco and/or alcohol use, whereas human papillomavirus (HPV) infection, particularly HPV16 infection, is increasingly recognized as a cause of oropharyngeal HNSCC. Despite clinical, histologic, and molecular differences between HPV-positive and HPV-negative HNSCCs, current treatment approaches are the same. For recurrent disease, these strategies include chemotherapy, immunotherapy with PD-1-inhibitors, or a monoclonal antibody, cetuximab, that targets epidermal growth factor; these therapies can be administered either as single agents or in combination. However, these treatment strategies carry a high risk of toxic side effects; therefore, more effective and less toxic treatments are needed. The landscape of HNSCC therapy is changing significantly; numerous clinical trials are underway to test novel therapeutic options like adaptive cellular therapy, antibody-drug conjugates, new targeted therapy agents, novel immunotherapy combinations, and therapeutic vaccines. This review helps in understanding the various developments in HNSCC therapy and sheds light on the path ahead in terms of further research in this field.

FDA Approval Summary: Olaparib Monotherapy or in Combination with Bevacizumab for the Maintenance Treatment of Patients with Advanced Ovarian Cancer.

On December 19, 2018, the U.S. Food and Drug Administration (FDA) granted approval to olaparib monotherapy for first-line maintenance treatment of BRCA-mutated (BRCAm) advanced ovarian cancer and, on May 8, 2020, expanded the indication of olaparib to include its use in combination with bevacizumab for first-line maintenance treatment of homologous recombination deficient (HRD)-positive advanced ovarian cancer. Both these approvals were based on randomized, double-blind, placebo-controlled trials. Approval for olaparib monotherapy was based on the SOLO-1 trial, comparing the efficacy of olaparib versus placebo in patients with BRCAm advanced ovarian, fallopian tube, or primary peritoneal cancer after surgical cytoreduction and first-line platinum-based chemotherapy. Two companion diagnostic (CDx) tests were approved with this indication: BRACAnalysis CDx, for germline BRCA1/2 alterations, and FoundationOne CDx, for BRCA1/2 alterations in tissue specimens. Approval for olaparib in combination with bevacizumab was based on the results of the PAOLA-1 trial that compared olaparib with bevacizumab versus placebo plus bevacizumab in patients with advanced high-grade epithelial ovarian cancer, fallopian tube, or primary peritoneal cancer after first-line platinum-based chemotherapy and bevacizumab. Myriad myChoice CDx was designated as a companion diagnostic device for use of olaparib plus bevacizumab combination for ovarian cancer associated with HRD-positive status. Both trials demonstrated clinically meaningful improvements in progression-free survival and favorable benefit-risk profiles for the indicated populations. This article summarizes the FDA thought process and data supporting the approval of olaparib as monotherapy and in combination with bevacizumab for maintenance therapy in this setting. IMPLICATIONS FOR PRACTICE: These approvals represent the first poly (ADP-ribose) polymerase inhibitor, alone or in combination with bevacizumab, approved in first-line maintenance treatment of women with advanced ovarian cancer after cytoreductive surgery and chemotherapy. In patients with BRCA-mutated tumors, olaparib monotherapy demonstrated a 70% reduction in the risk of disease progression or death compared with placebo, and olaparib in combination with bevacizumab demonstrated a 67% reduction in the risk of disease progression or death compared with bevacizumab alone in homologous recombination deficient-positive tumors. These approvals represent a major advance for the treatment of women with advanced ovarian cancer who are in complete or partial response after their initial platinum-based chemotherapy.

FDA Approval Summary: Rucaparib for the Treatment of Patients with Deleterious BRCA-Mutated Metastatic Castrate-Resistant Prostate Cancer.

The U.S. Food and Drug Administration (FDA) granted accelerated approval to rucaparib in May 2020 for the treatment of adult patients with deleterious BRCA mutation (germline and/or somatic)-associated metastatic castrate-resistant prostate cancer (mCRPC) who have been treated with androgen receptor-directed therapy and a taxane. This approval was based on data from the ongoing multicenter, open-label single-arm trial TRITON2. The primary endpoint, confirmed objective response rate, in the 62 patients who met the above criteria, was 44% (95% confidence interval [CI]: 31%-57%). The median duration of response was not estimable (95% CI: 6.4 to not estimable). Fifty-six percent of patients had a response duration of >6 months and 15% >12 months. The safety profile of rucaparib was generally consistent with that of the class of poly-(ADP-ribose) polymerase enzyme inhibitors and other trials of rucaparib in the treatment of ovarian cancer. Deaths due to adverse events (AEs) occurred in 1.7% of patients, and 8% discontinued rucaparib because of an AE. Grade 3-4 AEs occurred in 59% of patients. No patients with prostate cancer developed myelodysplastic syndrome or acute myeloid leukemia. The trial TRITON3 in patients with mCRPC is ongoing and is planned to verify the clinical benefit of rucaparib in mCRPC. This article summarizes the FDA thought process and data supporting this accelerated approval. IMPLICATIONS FOR PRACTICE: The accelerated approval of rucaparib for the treatment of adult patients with deleterious BRCA mutation (germline and/or somatic)-associated metastatic castrate-resistant prostate cancer who have been treated with androgen receptor-directed therapy and a taxane represents the first approved therapy for this selected patient population. This approval was based on a single-arm trial demonstrating a confirmed objective response rate greater than that of available therapy with a favorable duration of response and an acceptable toxicity profile. The ongoing trial TRITON3 is verifying the clinical benefit of this drug.

Structural diversity, functional aspects and future therapeutic applications of human gut microbiome.

The research on human gut microbiome, regarded as the black box of the human body, is still at the stage of infancy as the functional properties of the complex gut microbiome have not yet been understood. Ongoing metagenomic studies have deciphered that the predominant microbial communities belong to eubacterial phyla Firmicutes, Bacteroidetes, Proteobacteria, Fusobacteria, Cyanobacteria, Verrucomicrobia and archaebacterial phylum Euryarchaeota. The indigenous commensal microbial flora prevents opportunistic pathogenic infection and play undeniable roles in digestion, metabolite and signaling molecule production and controlling host's cellular health, immunity and neuropsychiatric behavior. Besides maintaining intestinal health via short-chain fatty acid (SCFA) production, gut microbes also aid in neuro-immuno-endocrine modulatory molecule production, immune cell differentiation and glucose and lipid metabolism. Interdependence of diet and intestinal microbial diversity suggests the effectiveness of pre- and pro-biotics in maintenance of gut and systemic health. Several companies worldwide have started potentially exploiting the microbial contribution to human health and have translated their use in disease management and therapeutic applications. The present review discusses the vast diversity of microorganisms playing intricate roles in human metabolism. The contribution of the intestinal microbiota to regulate systemic activities including gut-brain-immunity crosstalk has been focused. To the best of our knowledge, this review is the first of its kind to collate and discuss the companies worldwide translating the multi-therapeutic potential of human intestinal microbiota, based on the multi-omics studies, i.e. metagenomics and metabolomics, as ready solutions for several metabolic and systemic disorders.

Delineating Calcium Signaling Machinery in Plants: Tapping the Potential through Functional Genomics.

Plants have developed calcium (Ca2+) signaling as an important mechanism of  regulation of  stress perception,  developmental cues, and  responsive gene  expression. The  post-genomic era has witnessed the successful unravelling of the functional characterization of genes and the creation of large datasets of molecular information. The major elements of Ca2+ signaling machinery include Ca2+ sensors and responders such as Calmodulins (CaMs), Calmodulin-like proteins (CMLs), Ca2+/CaM-dependent protein kinases (CCaMKs), Ca2+-dependent protein kinases (CDPKs), Calcineurin B-like proteins (CBLs) as well as transporters, such as Cyclic nucleotide-gated channels (CNGCs), Glutamate-like receptors (GLRs), Ca2+-ATPases, Ca2+/H+ exchangers (CAXs) and mechanosensitive channels. These elements play an important role in the regulation of physiological processes and plant responses to various stresses. Detailed genomic analysis can help us in the identification of potential molecular targets that can be exploited towards the development of stress-tolerant crops. The information sourced from model systems through omics approaches helps in the prediction and simulation of regulatory networks involved in responses to different stimuli at the molecular and cellular levels. The molecular delineation of Ca2+ signaling pathways could be a stepping stone for engineering climate-resilient crop plants. Here, we review the recent developments in Ca2+ signaling in the context of transport, responses, and adaptations significant for crop improvement through functional genomics approaches.

Molecular and taxonomic characterization of arsenic (As) transforming Bacillus sp. strain IIIJ3-1 isolated from As-contaminated groundwater of Brahmaputra river basin, India.

BACKGROUND: Microbe-mediated redox transformation of arsenic (As) leading to its mobilization has become a serious environmental concern in various subsurface ecosystems especially within the alluvial aquifers. However, detailed taxonomic and eco-physiological attributes of indigenous bacteria from As impacted aquifer of Brahmaputra river basin has remained under-studied. RESULTS: A newly isolated As-resistant and -transforming facultative anaerobic bacterium IIIJ3-1 from As-contaminated groundwater of Jorhat, Assam was characterized. Near complete 16S rRNA gene sequence affiliated the strain IIIJ3-1 to the genus Bacillus and phylogenetically placed within members of B. cereus sensu lato group with B. cereus ATCC 14579(T) as its closest relative with a low DNA-DNA relatedness (49.9%). Presence of iC17:0, iC15:0 fatty acids and menaquinone 7 corroborated its affiliation with B. cereus group, but differential hydroxy-fatty acids, C18:2 and menaquinones 5 & 6 marked its distinctiveness. High As resistance [Maximum Tolerable Concentration = 10 mM As3+, 350 mM As5+], aerobic As3+ (5 mM) oxidation, and near complete dissimilatory reduction of As 5+ (1 mM) within 15 h of growth designated its physiological novelty. Besides O2, cells were found to reduce As5+, Fe3+, SO42-, NO3-, and Se6+ as alternate terminal electron acceptors (TEAs), sustaining its anaerobic growth. Lactate was the preferred carbon source for anaerobic growth of the bacterium with As5+ as TEA. Genes encoding As5+ respiratory reductase (arr A), As3+ oxidase (aioB), and As3+ efflux systems (ars B, acr3) were detected. All these As homeostasis genes showed their close phylogenetic lineages to Bacillus spp. Reduction in cell size following As exposure exhibited the strain's morphological response to toxic As, while the formation of As-rich electron opaque dots as evident from SEM-EDX possibly indicated a sequestration based As resistance strategy of strain IIIJ3-1. CONCLUSION: This is the first report on molecular, taxonomic, and ecophysiological characterization of a highly As resistant, As3+ oxidizing, and dissimilatory As5+ reducing Bacillus sp. IIIJ3-1 from As contaminated sites of Brahmaputra river basin. The strain's ability to resist and transform As along with its capability to sequester As within the cells demonstrate its potential in designing bioremediation strategies for As contaminated groundwater and other ecosystems.

Microcosm based analysis of arsenic release potential of Bacillus sp. strain IIIJ3-1 under varying redox conditions.

The role of indigenous bacteria in mobilization of sediment bound arsenic (As) into groundwater is investigated using subsurface sediment from Brahmaputra River Basin (BRB) and the Bacillus sp. strain IIIJ3-1, an indigenous species to BRB. Anaerobic sediment microcosms with varying organic carbon sources and terminal electron acceptors (TEAs) are used to illustrate the role of the test bacterium in As mobilization. The aquifer sediment shows an asymmetric distribution of As and Fe in its different phases. Among the TEAs added, NO3 amendment promotes higher cell growth, oxalic acid production and maximum release of sediment bound As. X-ray diffraction analysis further suggests that weathering of As bearing secondary minerals through bacterial action enhances As bioavailability, followed by dissimilatory reduction and thus promotes its mobilization into aqueous phase. Co-release pattern of other elements from the sediment indicates that release of As is decoupled from that of Fe. This study confirms that microbe-mediated mineral weathering followed by respiratory reduction of As facilitates mobilization of sediment hosted As into aqueous phase, and provides a better insight into the catabolic ability of groundwater bacteria in mobilization of sediment hosted As in BRB region.

Inhibition of a pancreatic cancer model by cooperative pairs of clinically approved and experimental antibodies.

By year 2025 pancreatic ductal adenocarcinoma (PDAC) is expected to become the second leading cause of cancer related death. However, other than improved chemotherapy and a small molecule inhibitor of the epidermal growth factor receptor (EGFR), no targeted drugs are currently available. Repurposing of approved drugs might offer a rapid solution. We employed an animal PDAC model, expressing a mutant and a wild type form of p53 and KRAS, respectively. Cetuximab, a clinically approved anti-EGFR monoclonal antibody (mAb) weakly inhibited PDAC xenografts, similar to trastuzumab, a mAb against HER2, a co-receptor of EGFR. Because the combination of cetuximab and trastuzumab only moderately enhanced the anti-tumor effects, we combined each with a home-made mAb to the same receptor and identified two cooperative pairs. The pair of trastuzumab and a murine anti-HER2 mAb better than the anti-EGFR pair inhibited PDAC xenografts, although HER2's abundance in our model is 15-fold lower than the level of EGFR. In vitro studies attribute cooperation to forced receptor endocytosis/degradation and inhibition of both DNA synthesis and cell migration. Taken together, our results identify cooperative pairs of anti-PDAC antibodies and highlight potential mechanisms of anti-tumor effects.

Enrichment of indigenous arsenate reducing anaerobic bacteria from arsenic rich aquifer sediment of Brahmaputra river basin and their potential role in as mobilization.

Anaerobic enrichment of As5+ reducing bacteria in the presence and/or absence of organic carbon (OC) and As5+ from As contaminated soil of Brahmaputra river basin (BRB) (Jorhat, Assam) was performed. Denaturing gradient gel electrophoresis of the 16SrRNA gene sequences amplified from the enriched microbial community indicated occurrence of maximum diversity under conditions receiving no OC (MSM) followed by moderate OC (LB). However, higher OC or As showed antagonistic effect on bacterial enrichment whereas together (BB + As) they showed a synergistic effect. Phylogenetic analysis of the prominent bands revealed an overall abundance of Lachnoanaerobaculum (39%), Clostridium (39%), Bacillus, Peptostreptococcaceae, Anaerostipes (13%), and Desulfotomaculum (8.7%). Moderate OC (LB) led to maximum As mobilization i.e. 27.42 µg/L, whereas presence of added As together with high OC (BB + As) enhanced the mobilization process. Mineralogical analyses of the sediments after incubation showed prominent weathering and loss of crystallinity in MSM and LB. Appearance of a new peak corresponding to arsenolamprite (As) in LB and LB + As indicated opening up of secondary phases of the minerals harboring As due to microbial leaching under moderate OC. This is the first study reporting Lachnoanaerobaculumas a potent As5+ dissimilating bacterium isolated from As contaminated subsurface sediment of BRB.

Iron Limitation Triggers Early Egress by the Intracellular Bacterial Pathogen Legionella pneumophila.

Legionella pneumophila is an intracellular bacterial pathogen that replicates in alveolar macrophages, causing a severe form of pneumonia. Intracellular growth of the bacterium depends on its ability to sequester iron from the host cell. In the L. pneumophila strain 130b, one mechanism used to acquire this essential nutrient is the siderophore legiobactin. Iron-bound legiobactin is imported by the transport protein LbtU. Here, we describe the role of LbtP, a paralog of LbtU, in iron acquisition in the L. pneumophila strain Philadelphia-1. Similar to LbtU, LbtP is a siderophore transport protein and is required for robust growth under iron-limiting conditions. Despite their similar functions, however, LbtU and LbtP do not contribute equally to iron acquisition. The Philadelphia-1 strain lacking LbtP is more sensitive to iron deprivation in vitro Moreover, LbtP is important for L. pneumophila growth within macrophages while LbtU is dispensable. These results demonstrate that LbtP plays a dominant role over LbtU in iron acquisition. In contrast, loss of both LbtP and LbtU does not impair L. pneumophila growth in the amoebal host Acanthamoeba castellanii, demonstrating a host-specific requirement for the activities of these two transporters in iron acquisition. The growth defect of the ΔlbtP mutant in macrophages is not due to alterations in growth kinetics. Instead, the absence of LbtP limits L. pneumophila replication and causes bacteria to prematurely exit the host cell. These results demonstrate the existence of a preprogrammed exit strategy in response to iron limitation that allows L. pneumophila to abandon the host cell when nutrients are exhausted.

Mechanism of Iron-Dependent Repressor (IdeR) Activation and DNA Binding: A Molecular Dynamics and Protein Structure Network Study.

Metalloproteins form a major class of enzymes in the living system that are involved in crucial biological functions such as catalysis, redox reactions and as 'switches' in signal transductions. Iron dependent repressor (IdeR) is a metal-sensing transcription factor that regulates free iron concentration in Mycobacterium tuberculosis. IdeR is also known to promote bacterial virulence, making it an important target in the field of therapeutics. Mechanistic details of how iron ions modulate IdeR such that it dimerizes and binds to DNA is not understood clearly. In this study, we have performed molecular dynamic simulations and integrated it with protein structure networks to study the influence of iron on IdeR structure and function. A significant structural variation between the metallated and the non-metallated system is observed. Our simulations clearly indicate the importance of iron in stabilizing its monomeric subunit, which in turn promotes dimerization. However, the most striking results are obtained from the simulations of IdeR-DNA complex in the absence of metals, where at the end of 100ns simulations, the protein subunits are seen to rapidly dissociate away from the DNA, thereby forming an excellent resource to investigate the mechanism of DNA binding. We have also investigated the role of iron as an allosteric regulator of IdeR that positively induces IdeR-DNA complex formation. Based on this study, a mechanistic model of IdeR activation and DNA binding has been proposed.

Genome annotation by shotgun inactivation of a native gene in hemizygous cells: application to BRCA2 with implication of hypomorphic variants.

The greatest interpretive challenge of modern medicine may be to functionally annotate the vast variation of human genomes. Demonstrating a proposed approach, we created a library of BRCA2 exon 27 shotgun-mutant plasmids including solitary and multiplex mutations to generate human knockin clones using homologous recombination. This 55-mutation, 13-clone syngeneic variance library (SyVaL) comprised severely affected clones having early-stop nonsense mutations, functionally hypomorphic clones having multiple missense mutations emphasizing the potential to identify and assess hypomorphic mutations in novel proteomic and epidemiologic studies, and neutral clones having multiple missense mutations. Efficient coverage of nonessential amino acids was provided by mutation multiplexing. Severe mutations were distinguished from hypomorphic or neutral changes by chemosensitivity assays (hypersensitivity to mitomycin C and acetaldehyde), by analysis of RAD51 focus formation, and by mitotic multipolarity. A multiplex unbiased approach of generating all-human SyVaLs in medically important genes, with random mutations in native genes, would provide databases of variants that could be functionally annotated without concerns arising from exogenous cDNA constructs or interspecies interactions, as a basis for subsequent proteomic domain mapping or clinical calibration if desired. Such gene-irrelevant approaches could be scaled up for multiple genes of clinical interest, providing distributable cellular libraries linked to public-shared functional databases.

Hypersensitivities for acetaldehyde and other agents among cancer cells null for clinically relevant Fanconi anemia genes.

Large-magnitude numerical distinctions (>10-fold) among drug responses of genetically contrasting cancers were crucial for guiding the development of some targeted therapies. Similar strategies brought epidemiological clues and prevention goals for genetic diseases. Such numerical guides, however, were incomplete or low magnitude for Fanconi anemia pathway (FANC) gene mutations relevant to cancer in FANC-mutation carriers (heterozygotes). We generated a four-gene FANC-null cancer panel, including the engineering of new PALB2/FANCN-null cancer cells by homologous recombination. A characteristic matching of FANCC-null, FANCG-null, BRCA2/FANCD1-null, and PALB2/FANCN-null phenotypes was confirmed by uniform tumor regression on single-dose cross-linker therapy in mice and by shared chemical hypersensitivities to various inter-strand cross-linking agents and γ-radiation in vitro. Some compounds, however, had contrasting magnitudes of sensitivity; a strikingly high (19- to 22-fold) hypersensitivity was seen among PALB2-null and BRCA2-null cells for the ethanol metabolite, acetaldehyde, associated with widespread chromosomal breakage at a concentration not producing breaks in parental cells. Because FANC-defective cancer cells can share or differ in their chemical sensitivities, patterns of selective hypersensitivity hold implications for the evolutionary understanding of this pathway. Clinical decisions for cancer-relevant prevention and management of FANC-mutation carriers could be modified by expanded studies of high-magnitude sensitivities.

Connexin-43 regulates p38-mediated cell migration and invasion induced selectively in tumour cells by low doses of γ-radiation in an ERK-1/2-independent manner.

Radiotherapy exposes certain regions of solid tumours to low sublethal doses of γ-radiation that may cause secondary malignancies. Therefore, evaluating low-dose-γ-radiation-induced alterations in tumorigenic potential and understanding their mechanisms could help in improving radiotherapy outcome. Limited studies have indicated connexin (Cx) up-regulation by low doses, whereas Cxs are independently shown to alter cell migration in unirradiated cells. We investigated low-dose-γ-radiation-induced alterations in Cx43 expression and cell proliferation/migration/invasion in various tumour cell lines, along with the putative molecular pathways such as p38 and extracellular signal-regulated kinase-1/2 (ERK-1/2)-mitogen-activated protein kinases (MAPKs). Interestingly, a narrow range of low doses (10-20 cGy) enhanced Cx43 expression and also selectively induced glioma cell migration without altering cell proliferation, accompanied by sustained activation of p38 and up-regulation of p21(waf1/cip1), whereas the lowest (5 cGy) dose induced cell proliferation coupled with enhanced p-ERK1/2, proliferating cell nuclear antigen and p-H3 levels without inducing cell migration. Most importantly, low-dose-γ-radiation-induced cell migration and p38 activation was strongly inhibited by knocking down Cx43 expression, thereby demonstrating latter's upstream role, whereas the knock-down had no effect on ERK-1/2 or cell proliferation. Silencing Cx43 caused near-complete inhibition of radiation-induced cell migration/invasion in all tumour cell lines (U87, BMG-1, A549 and HeLa), whereas no cell migration/invasiveness was induced in the γ-irradiated primary VH10 or transformed AA8 fibroblasts. Our study demonstrates for the first time that low-dose γ-radiation induces p38-MAPK mediated cell migration selectively in tumour cells. Further, this effect is regulated by Cx43, which could thus be an important mediator in radiation-induced secondary malignancies and/or metastasis.