Human haplotype block sizes are negatively correlated with recombination rates

The International Haplotype Map ("HapMap") Project is motivated, in part, by the belief that the organization of the human genome, the mechanics of recombination, and the population-level behavior of alleles at adjacent loci should allow researchers to parse the genome into small segments, or "blocks," that show strong linkage disequilibrium (LD) between alleles at loci within those segments. The discovery and evidence for these blocks is to be based solely on the observed LD strength and patterns between alleles at adjacent loci throughout the genome. Although there are many factors that contribute to LD strength, we assessed the correlation between block structure, in terms of length and percentage of the genome assembled into blocks within a region, and recombination rate obtained from two independent sources. We found evidence of a striking negative correlation between the average recombination rate and average block length, suggesting that recombination rate is a strong contributor to haplotype block structure within the genome. We discuss the potential implications of this negative correlation in the context of the organization, properties, and potential ubiquity of a block-like structure in the human genome.     

Carcinogenic polycyclic aromatic hydrocarbon-DNA adducts and mechanism of action

Polycyclic aromatic hydrocarbons (PAHs) are a class of widespread environmental carcinogens. Most of our knowledge of their mechanisms of metabolic activation to DNA-binding "ultimate carcinogenic" metabolites has come from analysis of the DNA interaction products formed by these highly reactive intermediates. Studies of their role in forming DNA-binding intermediates identical to those formed in vivo from the PAH itself have also allowed identification of the particular cytochrome P450 enzymes involved in activating various structural classes of carcinogenic PAHs. It has been established that PAHs, after metabolic activation in vivo, are capable of inducing mutations in oncogenes and, by inducing multiple mutations, may result in tumors. PAHs also cause changes in cellular gap-junction communication similar to those caused by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. Thus, PAHs may also act through a promotional mechanism in addition to serving as tumor initiators. Previous studies on these mechanisms are described and summarized.     

Impaired fertility caused by endocrine dysfunction in women.

Infertility can be frustrating for patients and physicians. Endocrine dysfunction can impair fertility and alter pregnancy outcome. Once the diagnosis is secured, most endocrine disorders are reversible or can be adequately managed to restore fertility and decrease associated pregnancy complications. Improved understanding of the subtleties and intricacies of mechanisms by which endocrinopathies can hinder fertility and influence the course of a pregnancy is vital, more so in the era of assisted reproductive technology, because pregnancy in an abnormal endocrine environment can be disastrous. The role of autoimmunity in infertility and pregnancy loss is less clear; in this context, there are limited management options to improve fertility and overall pregnancy outcomes.     

Clinical pertinence of neutrophil-to- lymphocyte ratio among hypertensives with different grades and duration of hypertension – an insight

Over the recent years, the pathophysiology of the inflammatory component in hypertension has been a challenge, because this inflammatory response is mainly contributed by an increased oxidative stress with the release of inflammatory mediators. Identification of a simple and early inflammatory marker such as the neutrophil-to-lymphocyte ratio (NLR) is the need of the hour. This study correlates the same specifically taking into account the duration and the grades of hypertension.

Objective: The response of the NLR among the hypertensives and its correlation with duration and stages of hypertension.

Method: Totally, 80 subjects and 40 controls of age between 20 and 60 years and both genders were included. Three recordings of blood pressure were measured with a standard mercury sphygmomanometer. The differential leukocyte count was estimated with an automated Beckman Coulter.

Objective: Variations in the neutrophil and lymphocyte counts were significant among the hypertensives with a p-value < 0.001. The NLR was also significantly altered among the hypertensives with a p-value = 0.001. The NLR showed a rise in value among the normotensives, prehypertensives, and stage 1 of systolic hypertension, though not statistically significant. An increase in the NLR was observed in hypertensives with duration of 1–2 years.

Conclusion: Our study gives a new insight with a rise in NLR in early years and even among prehypertensives and stage 1 systolic hypertension under strict criterion. This could be utilized as an early predictive tool, relating the inflammatory process and hypertension which on further intervention could slow the progression of the disease process.

Abbreviations: NLR: Neutrophil-to-lymphocyte ratio; BP: Blood pressure.     

Oncogenic KRAS engages an RSK1/NF1 pathway to inhibit wild-type RAS signaling in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy with limited treatment options. Although activating mutations of the KRAS GTPase are the predominant dependency present in >90% of PDAC patients, targeting KRAS mutants directly has been challenging in PDAC. Similarly, strategies targeting known KRAS downstream effectors have had limited clinical success due to feedback mechanisms, alternate pathways, and dose-limiting toxicities in normal tissues. Therefore, identifying additional functionally relevant KRAS interactions in PDAC may allow for a better understanding of feedback mechanisms and unveil potential therapeutic targets. Here, we used proximity labeling to identify protein interactors of active KRAS in PDAC cells. We expressed fusions of wild-type (WT) (BirA-KRAS4B), mutant (BirA-KRAS4B^G12D), and nontransforming cytosolic double mutant (BirA-KRAS4B^G12D/C185S) KRAS with the BirA biotin ligase in murine PDAC cells. Mass spectrometry analysis revealed that RSK1 selectively interacts with membrane-bound KRAS^G12D, and we demonstrate that this interaction requires NF1 and SPRED2. We find that membrane RSK1 mediates negative feedback on WT RAS signaling and impedes the proliferation of pancreatic cancer cells upon the ablation of mutant KRAS. Our findings link NF1 to the membrane-localized functions of RSK1 and highlight a role for WT RAS signaling in promoting adaptive resistance to mutant KRAS-specific inhibitors in PDAC.

A total of 60,430 new cases of pancreatic cancer were estimated for 2021, and the 5-y relative survival rate has consistently remained below 11% (1). About 85% of these pancreatic cancer tumors are pancreatic ductal adenocarcinoma (PDAC) (2). Poor outcomes of PDAC cases result from late diagnoses leading to unresectable and heterogeneous tumors as well as ineffective therapies, which only prolong survival on the order of months (3–5). Mutations in the KRAS proto-oncogene are present in over 90% of PDAC cases and are associated with a poor prognosis (6). Furthermore, mice expressing mutant KRAS in the pancreas develop precursor lesions, which sporadically progress into frank PDAC. This progression is accelerated when combined with other mutations or deletion of tumor suppressor genes (7–11). Additionally, independent studies have shown that the maintenance of murine PDAC cells require KRAS (12–14).As a RAS GTPase, KRAS acts as a molecular switch at the plasma membrane that relays growth factor signaling from receptor tyrosine kinases to downstream pathways such as RAF/MEK and PI3K/AKT (15). GTP binding alters the conformation of the KRAS G domain, thereby creating binding sites for downstream effectors to trigger enzymatic cascades that promote cell transformation (16–19). Intrinsically, KRAS slowly hydrolyzes GTP into GDP to halt signaling; however, GTPase activating proteins (GAPs) such as neurofibromin 1(NF1) catalyze this process (20). In contrast, guanine nucleotide exchange factors, such as son of sevenless homolog 1 (SOS1), catalyze the exchange of GTP for bound GDP. In most PDAC cases, KRAS is mutated at the 12th residue located in the G domain from glycine to either a valine (G12V), or more commonly, aspartate (G12D). These mutations sterically prevent the “arginine finger domain” of GAPs from entering the GTPase site, thereby blocking extrinsic allosteric GTPase activation and stabilizing RAS-GTP (21, 22). Activating mutations in KRAS constitutively trigger RAF/MEK and PI3K/AKT pathways leading to increased cell proliferation as well as other prooncogenic behaviors (15). KRAS signaling not only relies on the G domain but also the C-terminal hypervariable domain (HVR), which is required to stabilize KRAS on membranes where signaling is most efficient (23–26). Independent studies suggest that specific biochemical and cellular consequences of KRAS activation are attributed to the unique properties of the HVR of the predominant splice form KRAS4B, namely the polybasic domain and the lipid anchor (27–30). Localization of RAS proteins to the plasma membrane requires the prenylation of the CAAX motif (23). Additionally, for KRAS4B, the hypervariable region contains a highly polybasic domain consisting of several consecutive lysines, which can interact with the negative charges on the polar heads of phospholipids and stabilize protein interactions (31). Structural and biochemical characterization of the HVR and G domain has contributed to a better understanding of the signaling outputs of KRAS and led to KRAS-targeting strategies.Various approaches to inhibit KRAS include direct inhibition, expression interference, mislocalization, and targeting of downstream effectors (32). Thus far, direct inhibitors against KRAS have only successfully targeted the G12C mutant, which comprises 2.9% of KRAS mutant PDAC (21, 33). For other KRAS mutants, targeting downstream effectors of KRAS in pancreatic cancer remains an alternative approach. Unfortunately, dual inhibition of MEK and AKT pathways was ineffective in PDAC patients (34). Difficulty in targeting KRAS due to adaptive resistance and feedback regulation motivates a better understanding of KRAS biology (35). For example, although PDAC typically features a mutant KRAS, there may be a role for its wild-type (WT) counterpart as well as WT RAS paralogs (HRAS and NRAS), which are GAP sensitive and subject to signaling feedback. While oncogenic KRAS has been shown to activate WT HRAS and NRAS via allosteric stimulation of SOS1 (36), WT KRAS has been proposed to be a tumor suppressor in some KRAS mutant cancers based on the commonly observed mutant-specific allele imbalance that occurs throughout tumor progression (37). Additionally, the reintroduction of WT KRAS abolished tumor T cell acute lymphoblastic leukemia development and impaired tumor growth in KRAS mutant lung cancer cells in vivo (37–39). The discovery of novel KRAS protein interactors involved in downstream signaling or feedback and compensatory pathways may elucidate why inhibition of downstream pathways have had limited clinical impact in PDAC. Here, we perform proximity labeling experiments by expressing a fusion of BirA^R118G biotin ligase and KRAS in PDAC cells, which, in the presence of high concentrations of biotin, generates reactive biotinoyl-AMP that labels lysines of nearby proteins, such as interactors of its fusion partner KRAS (40–42). The biotinylated interactor proteins can be isolated by streptavidin pulldown and analyzed by proteomics to identify novel protein interactors (43–45). Because covalent labeling occurs in living cells, enzymatic labeling may potentially identify transient interactors and protein complexes.Two recent studies used proximity-dependent biotin identification (BioID) labeling methods to identify KRAS interactors in 293T and colon cancer cells (46, 47). These studies uncovered and validated the functional relevance of PIP5KA1 and mTORC2 in PDAC cells. However, BirA-KRAS screens in PDAC models have not yet been performed. Since the tumor context may determine protein expression and relevant interactions, we sought to perform a BirA-KRAS screen in PDAC cells. We hypothesize that proximity labeling with BioID presents a means for identifying new mutant KRAS-specific interactions in PDAC, which may unveil new insights into therapeutic design for this malignancy.     

Relationship between T cell activation and CD4+ T cell count in HIV-seropositive individuals with undetectable plasma HIV RNA levels in the absence of therapy

BACKGROUND: Although untreated human immunodeficiency virus (HIV)-infected patients maintaining undetectable plasma HIV RNA levels (elite controllers) have high HIV-specific immune responses, it is unclear whether they experience abnormal levels of T cell activation, potentially contributing to immunodeficiency. METHODS: We compared percentages of activated (CD38(+)HLA-DR(+)) T cells between 30 elite controllers, 47 HIV-uninfected individuals, 187 HIV-infected individuals with undetectable viremia receiving antiretroviral therapy (antiretroviral therapy suppressed), and 66 untreated HIV-infected individuals with detectable viremia. Because mucosal translocation of bacterial products may contribute to T cell activation in HIV infection, we also measured plasma lipopolysaccharide (LPS) levels. RESULTS: Although the median CD4(+) cell count in controllers was 727 cells/mm(3), 3 (10%) had CD4(+) cell counts <350 cells/mm(3) and 2 (7%) had acquired immunodeficiency syndrome. Controllers had higher CD4(+) and CD8(+) cell activation levels (P < .001 for both) than HIV-negative subjects and higher CD8(+) cell activation levels than the antiretroviral therapy suppressed (P = .048). In controllers, higher CD4(+) and CD8(+) T cell activation was associated with lower CD4(+) cell counts (P = .009 and P = .047). Controllers had higher LPS levels than HIV-negative subjects (P < .001), and in controllers higher LPS level was associated with higher CD8(+) T cell activation (P = .039). CONCLUSION: HIV controllers have abnormally high T cell activation levels, which may contribute to progressive CD4(+) T cell loss even without measurable viremia.     

Loss of T cell responses following long-term cryopreservation

Although cryopreservation of peripheral blood mononuclear cells (PBMC) is a commonly used technique, the degree to which it affects subsequent functional studies has not been well defined. Here we demonstrate that long-term cryopreservation has detrimental effects on T cell IFN-gamma responses in human immunodeficiency virus (HIV) infected individuals. Long-term cryopreservation caused marked decreases in CD4(+) T cell responses to whole proteins (HIV p55 and cytomegalovirus (CMV) lysate) and HIV peptides, and more limited decreases in CD8(+) T cell responses to whole proteins. These losses were more apparent in cells stored for greater than one year compared to less than six months. CD8(+) T cell responses to peptides and peptide pools were well preserved. Loss of both CD4(+) and CD8(+) T cell responses to CMV peptide pools were minimal in HIV-negative individuals. Addition of exogenous antigen presenting cells (APC) did not restore CD4(+) T cell responses to peptide stimulation and partially restored T cell IFN-gamma responses to p55 protein. Overnight resting of thawed cells did not restore T cell IFN-gamma responses to peptide or whole protein stimulation. A selective loss of phenotypically defined effector cells did not explain the decrement of responses, although cryopreservation did increase CD4(+) T cell apoptosis, possibly contributing to the loss of responses. These data suggest that the impact of cryopreservation should be carefully considered in future vaccine and pathogenesis studies. In HIV-infected individuals short-term cryopreservation may be acceptable for measuring CD4(+) and CD8(+) T cell responses. Long-term cryopreservation, however, may lead to the loss of CD4(+) T cell responses and mild skewing of T cell phenotypic marker expression.