Characterization of a Ferryl Flip in Electronically Tuned Nonheme Complexes. Consequences in Hydrogen Atom Transfer Reactivity.

Two oxoiron(IV) isomers (R 2a and R 2b) of general formula [FeIV (O)(R PyNMe3 )(CH3 CN)]2+ are obtained by reaction of their iron(II) precursor with NBu4 IO4 . The two isomers differ in the position of the oxo ligand, cis and trans to the pyridine donor. The mechanism of isomerization between R 2a and R 2b has been determined by kinetic and computational analyses uncovering an unprecedented path for interconversion of geometrical oxoiron(IV) isomers. The activity of the two oxoiron(IV) isomers in hydrogen atom transfer (HAT) reactions shows that R 2a reacts one order of magnitude faster than R 2b, which is explained by a repulsive noncovalent interaction between the ligand and the substrate in R 2b. Interestingly, the electronic properties of the R substituent in the ligand pyridine ring do not have a significant effect on reaction rates. Overall, the intrinsic structural aspects of each isomer define their relative HAT reactivity, overcoming changes in electronic properties of the ligand.

Dynamics and determinants of the force of infection of dengue virus from 1994 to 2015 in Managua, Nicaragua.

Dengue virus (DENV) is the most prevalent human vector-borne viral disease. The force of infection (FoI), the rate at which susceptible individuals are infected in a population, is an important metric for infectious disease modeling. Understanding how and why the FoI of DENV changes over time is critical for developing immunization and vector control policies. We used age-stratified seroprevalence data from 12 years of the Pediatric Dengue Cohort Study in Nicaragua to estimate the annual FoI of DENV from 1994 to 2015. Seroprevalence data revealed a change in the rate at which children acquire DENV-specific immunity: in 2004, 50% of children age >4 years were seropositive, but by 2015, 50% seropositivity was reached only by age 11 years. We estimated a spike in the FoI in 1997-1998 and 1998-1999 and a gradual decline thereafter, and children age <4 years experienced a lower FoI. Two hypotheses to explain the change in the FoI were tested: (i) a transition from introduction of specific DENV serotypes to their endemic transmission and (ii) a population demographic transition due to declining birth rates and increasing life expectancy. We used mathematical models to simulate these hypotheses. We show that the initial high FoI can be explained by the introduction of DENV-3 in 1994-1998, and that the overall gradual decline in the FoI can be attributed to demographic shifts. Changes in immunity and demographics strongly impacted DENV transmission in Nicaragua. Population-level measures of transmission intensity are dynamic and thus challenging to use to guide vaccine implementation locally and globally.

Granulocyte-monocyte apheresis: an alternative combination therapy after loss of response to anti-TNF agents in ulcerative colitis.

Objective: To evaluate the effectiveness and safety of the combination of granulocyte-monocyte apheresis (GMA) after loss of response (LOR) to anti-tumor necrosis factor (TNF) agents in ulcerative colitis (UC). Materials and methods: A retrospective, multicenter study was performed in 11 inflammatory bowel disease (IBD) Units. Clinical remission was defined as a partial Mayo score ≤2. The effectiveness of the treatment was evaluated by the partial Mayo score and the rate of anti-TNF intensification, switch, swap or colectomy. Results: Forty-seven patients with ulcerative colitis were included (mean age 35 years, mean disease duration 52 months, 66% male and 59% extensive colitis). Twenty-three subjects were receiving infliximab, eighteen adalimumab and six golimumab. GMA was combined after a primary non-response (49%) or secondary loss of response (51%) to anti-TNF therapy. We observed a significant decrease in partial Mayo score and fecal calprotectin after GMA. Fifteen patients (32%) responded to the combination therapy without anti-TNF intensification, switch, swap or colectomy. Eight patients (17%) underwent colectomy. Two patients (4%) presented adverse events related to the technique. Conclusions: Combination of GMA and anti-tumor necrosis factor is a safe and effective treatment after the loss of response to these biologic agents, with a significant decrease of the clinical disease activity and biomarkers, in a population with limited therapeutic alternatives.

PDGF-AB and 5-Azacytidine induce conversion of somatic cells into tissue-regenerative multipotent stem cells.

Current approaches in tissue engineering are geared toward generating tissue-specific stem cells. Given the complexity and heterogeneity of tissues, this approach has its limitations. An alternate approach is to induce terminally differentiated cells to dedifferentiate into multipotent proliferative cells with the capacity to regenerate all components of a damaged tissue, a phenomenon used by salamanders to regenerate limbs. 5-Azacytidine (AZA) is a nucleoside analog that is used to treat preleukemic and leukemic blood disorders. AZA is also known to induce cell plasticity. We hypothesized that AZA-induced cell plasticity occurs via a transient multipotent cell state and that concomitant exposure to a receptive growth factor might result in the expansion of a plastic and proliferative population of cells. To this end, we treated lineage-committed cells with AZA and screened a number of different growth factors with known activity in mesenchyme-derived tissues. Here, we report that transient treatment with AZA in combination with platelet-derived growth factor-AB converts primary somatic cells into tissue-regenerative multipotent stem (iMS) cells. iMS cells possess a distinct transcriptome, are immunosuppressive, and demonstrate long-term self-renewal, serial clonogenicity, and multigerm layer differentiation potential. Importantly, unlike mesenchymal stem cells, iMS cells contribute directly to in vivo tissue regeneration in a context-dependent manner and, unlike embryonic or pluripotent stem cells, do not form teratomas. Taken together, this vector-free method of generating iMS cells from primary terminally differentiated cells has significant scope for application in tissue regeneration.

Comparison of Four Serological Methods and Two Reverse Transcription-PCR Assays for Diagnosis and Surveillance of Zika Virus Infection.

Zika virus (ZIKV) is a mosquito-borne flavivirus that is responsible for recent explosive epidemics in the Americas. Notably, ZIKV infection during pregnancy has been found to cause congenital birth defects, including microcephaly, and ZIKV has been associated with Guillain-Barré syndrome in adults. Diagnosis and surveillance of Zika in the Americas have been challenging due to similar clinical manifestations and extensive antibody cross-reactivity with endemic flaviviral diseases, such as dengue. We evaluated four serological and two reverse transcription-PCR (RT-PCR) methods in acute-phase (mean day, 1.8), early-convalescent-phase (mean day, 16.7), and late-convalescent-phase (mean, ~7 months) samples from the same individuals in a long-term pediatric cohort study in Nicaragua. Well-characterized samples from 301 cases of Zika, dengue, or non-Zika, nondengue febrile illnesses were tested. Compared to a composite reference, an in-house IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) and the NIAID-Biodefense and Emerging Infections (BEI) MAC-ELISA measuring IgM yielded sensitivities of 94.5% and 70.1% and specificities of 85.6% and 82.8%, respectively. The NS1 blockade-of-binding ELISA measuring anti-ZIKV NS1 antibody levels yielded sensitivities of 85.0% and 96.5% and specificities of 91.4% and 92.6% at early and late convalescence, respectively. An inhibition ELISA detecting total anti-ZIKV antibodies had sensitivity and specificity values of 68.3% and 58.3% for diagnosis and 94.0% and 98.6% for measuring annual infection incidence. Finally, the ZCD and Trioplex real-time RT-PCR assays detecting Zika, chikungunya, and dengue viruses both yielded a sensitivity of 96.1% and specificity of 100%. Together, these assays resolve the urgent need for diagnostic and surveillance tools for countries affected by Zika virus infections.

A cryptic paracentric inversion of MSH2 exons 2-6 causes Lynch syndrome.

Lynch syndrome is an autosomal dominant disorder that predisposes carriers of DNA mismatch repair (MMR) gene mutations to early-onset cancer. Germline testing screens exons and splice sites for mutations, but does not examine introns or RNA transcripts for alterations. Pathogenic mutations have not been detected in ~30% of suspected Lynch syndrome cases with standard screening practices. We present a 38-year-old male with a clinicopathological and family history consistent with Lynch syndrome, including loss of MSH2 expression in his tumor. Germline testing revealed normal MSH2 coding sequence, splice sites and exon copy number, however, cDNA sequencing identified an aberrant MSH2 transcript lacking exons 2-6. An inversion PCR on germline DNA identified an ~18kb unbalanced, paracentric inversion within MSH2, with breakpoints in a long terminal repeat in intron 1 and an Alu repeat in intron 6. The 3' end of the inversion had a 1.2 kb deletion and an 8 bp insertion at the junction with intron 6. Screening of 55 additional Australian patients presenting with MSH2-deficient tumors who were negative in germline genetic tests for MSH2 mutations identified another inversion-positive patient. We propose an Alu-mediated recombination model to explain the origin of the inversion. Our study illustrates the potential value of cDNA screening to identify patients with cryptic MMR gene rearrangements, clarifies why standard testing may not detect some pathogenic alterations, and provides a genetic test for screening individuals with suspected Lynch syndrome that present with unexplained MSH2-deficient tumors.

Viremia and Clinical Presentation in Nicaraguan Patients Infected With Zika Virus, Chikungunya Virus, and Dengue Virus.

BACKGROUND: Zika virus (ZIKV), chikungunya virus (CHIKV), and dengue virus (DENV) cocirculate in Nicaragua. In this study, we sought to compare the quantified viremia and clinical presentation of patients infected with 1 or more of these viruses. METHODS: Acute-phase serum samples from 346 patients with a suspected arboviral illness were tested using a multiplex real-time reverse-transcription polymerase chain reaction for ZIKV, CHIKV, and DENV. Viremia was quantitated for each detected virus, and clinical information from request forms submitted with each sample was recorded. RESULTS: A total of 263 patients tested positive for 1 or more viruses: 192 patients tested positive for a single virus (monoinfections) and 71 patients tested positive for 2 or all 3 viruses (coinfections). Quantifiable viremia was lower in ZIKV infections compared with CHIKV or DENV (mean 4.70 vs 6.42 and 5.84 log10 copies/mL serum, respectively; P < .001 for both comparisons), and for each virus, mean viremia was significantly lower in coinfections than in monoinfections. Compared with patients with CHIKV or DENV, ZIKV patients were more likely to have a rash (P < .001) and less likely to be febrile (P < .05) or require hospitalization (P < .001). Among all patients, hospitalized cases had higher viremia than those who did not require hospitalization (7.1 vs 4.1 log10 copies/mL serum, respectively; P < .001). CONCLUSIONS: ZIKV, CHIKV, and DENV result in similar clinical presentations, and coinfections may be relatively common. Our findings illustrate the need for accurate, multiplex diagnostics for patient care and epidemiologic surveillance.

Reassembly of nucleosomes at the MLH1 promoter initiates resilencing following decitabine exposure.

Hypomethylating agents reactivate tumor suppressor genes that are epigenetically silenced in cancer. Inevitably these genes are resilenced, leading to drug resistance. Using the MLH1 tumor suppressor gene as a model, we showed that decitabine-induced re-expression was dependent upon demethylation and eviction of promoter nucleosomes. Following decitabine withdrawal, MLH1 was rapidly resilenced despite persistent promoter demethylation. Single molecule analysis at multiple time points showed that gene resilencing was initiated by nucleosome reassembly on demethylated DNA and only then was followed by remethylation and stable silencing. Taken together, these data establish the importance of nucleosome positioning in mediating resilencing of drug-induced gene reactivation and suggest a role for therapeutic targeting of nucleosome assembly as a mechanism to overcome drug resistance.

Lynch syndrome associated with two MLH1 promoter variants and allelic imbalance of MLH1 expression.

Lynch syndrome is a hereditary cancer syndrome caused by a constitutional mutation in one of the mismatch repair genes. The implementation of predictive testing and targeted preventative surveillance is hindered by the frequent finding of sequence variants of uncertain significance in these genes. We aimed to determine the pathogenicity of previously reported variants (c.-28A>G and c.-7C>T) within the MLH1 5'untranslated region (UTR) in two individuals from unrelated suspected Lynch syndrome families. We investigated whether these variants were associated with other pathogenic alterations using targeted high-throughput sequencing of the MLH1 locus. We also determined their relationship to gene expression and epigenetic alterations at the promoter. Sequencing revealed that the c.-28A>G and c.-7C>T variants were the only potentially pathogenic alterations within the MLH1 gene. In both individuals, the levels of transcription from the variant allele were reduced to 50% compared with the wild-type allele. Partial loss of expression occurred in the absence of constitutional epigenetic alterations within the MLH1 promoter. We propose that these variants may be pathogenic due to constitutional partial loss of MLH1 expression, and that this may be associated with intermediate penetrance of a Lynch syndrome phenotype. Our findings provide further evidence of the potential importance of noncoding variants in the MLH1 5'UTR in the pathogenesis of Lynch syndrome.

Exploring the genetic control of sweat gland characteristics in beef cattle for enhanced heat tolerance.

BACKGROUND: Thermal stress in subtropical regions is a major limiting factor in beef cattle production systems with around $369 million being lost annually due to reduced performance. Heat stress causes numerous physiological and behavioral disturbances including reduced feed intake and decreased production levels. Cattle utilize various physiological mechanisms such as sweating to regulate internal heat. Variation in these traits can help identify genetic variants that control sweat gland properties and subsequently allow for genetic selection of cattle with greater thermotolerance. METHODS: This study used 2,401 Brangus cattle from two commercial ranches in Florida. Precise phenotypes that contribute to an animal's ability to manage heat stress were calculated from skin biopsies and included sweat gland area, sweat gland depth, and sweat gland length. All animals were genotyped with the Bovine GGP F250K, and BLUPF90 software was used to estimate genetic parameters and for Genome Wide Association Study. RESULTS: Sweat gland phenotypes heritability ranged from 0.17 to 0.42 indicating a moderate amount of the phenotypic variation is due to genetics, allowing producers the ability to select for favorable sweat gland properties. A weighted single-step GWAS using sliding 10 kb windows identified multiple quantitative trait loci (QTLs) explaining a significant amount of genetic variation. QTLs located on BTA7 and BTA12 explained over 1.0% of genetic variance and overlap the ADGRV1 and CCDC168 genes, respectively. The variants identified in this study are implicated in processes related to immune function and cellular proliferation which could be relevant to heat management. Breed of Origin Alleles (BOA) were predicted using local ancestry in admixed populations (LAMP-LD), allowing for identification of markers' origin from either Brahman or Angus ancestry. A BOA GWAS was performed to identify regions inherited from particular ancestral breeds that might have a significant impact on sweat gland phenotypes. CONCLUSIONS: The results of the BOA GWAS indicate that both Brahman and Angus alleles contribute positively to sweat gland traits, as evidenced by favorable marker effects observed from both genetic backgrounds. Understanding and utilizing genetic traits that confer better heat tolerance is a proactive approach to managing the impacts of climate change on livestock farming.

Comparative Analysis of SARS-CoV-2 Antibody Responses across Global and Lesser-Studied Vaccines.

Few data are available on antibody response for some SARS-CoV-2 vaccines, and there is a lack of ability to compare vaccine responses in the same population. This cross-sectional study conducted in Nicaragua examines the SARS-CoV-2 antibody responses in individuals, previously exposed to high infection rates who have received various vaccines. The vaccines under comparison include well-known ones like Pfizer (BNT162b2) and AstraZeneca (ChAdOx1-S), alongside less-studied vaccines including Soberana (Soberana 02), Abdala (CIGB-66), and Sputnik V/Sputnik Light. Overall, 3195 individuals participated, with 2862 vaccinated and 333 unvaccinated. We found that 95% of the unvaccinated were seropositive, with much lower titers than the vaccinated. Among the vaccinated, we found that Soberana recipients mounted the highest anti-spike response (mean difference (MD) = 36,498.8 [20,312.2, 52,685.5]), followed by Abdala (MD = 25,889.9 [10,884.1, 40,895.7]), BNT162b2 (MD = 12,967.2 [7543.7, 18,390.8]) and Sputnik with AstraZeneca as the reference group, adjusting for age, sex, vaccine status, days after last dose, and self-reported COVID-19. In addition, we found that subjects with complete vaccination series had higher antibody magnitude than those with incomplete series. Overall, we found no evidence of waning in the antibody magnitude across vaccines. Our study supports the conclusion that populations with high infection rates still benefit substantially from vaccination.

Intramolecular C-H Oxidation in Iron(V)-oxo-carboxylato Species Relevant in the γ-Lactonization of Alkyl Carboxylic Acids.

High-valent oxoiron species have been invoked as oxidizing agents in a variety of iron-dependent oxygenases. Taking inspiration from nature, selected nonheme iron complexes have been developed as catalysts to elicit C-H oxidation through the mediation of putative oxoiron(V) species, akin to those proposed for Rieske oxygenases. The addition of carboxylic acids in these iron-catalyzed C-H oxidations has proved highly beneficial in terms of product yields and selectivities, suggesting the direct involvement of iron(V)-oxo-carboxylato species. When the carboxylic acid functionality is present in the alkane substrate, it acts as a directing group, enabling the selective intramolecular γ-C-H hydroxylation that eventually affords γ-lactones. While this mechanistic frame is solidly supported by previous mechanistic studies, direct spectroscopic detection of the key iron(V)-oxo-carboxylato intermediate and its competence for engaging in the selective γ-C-H oxidation leading to lactonization have not been accomplished. In this work, we generate a series of well-defined iron(V)-oxo-carboxylato species (2c-2f) differing in the nature of the bound carboxylate ligand. Species 2c-2f are characterized by a set of spectroscopic techniques, including UV-vis spectroscopy, cold-spray ionization mass spectrometry (CSI-MS), and, in selected cases, EPR and Mössbauer spectroscopies. We demonstrate that 2c-2f undergo site-selective γ-lactonization of the carboxylate ligand in a stereoretentive manner, thus unequivocally identifying metal-oxo-carboxylato species as the powerful yet selective C-H cleaving species in catalytic γ-lactonization reactions of carboxylic acids. Reactivity experiments confirm that the intramolecular formation of γ-lactones is in competition with the intermolecular oxidation of external alkanes and olefins. Finally, mechanistic studies, together with DFT calculations, support a mechanism involving a site-selective C-H cleavage in the γ-position of the carboxylate ligand by the oxo moiety, followed by a fast carboxylate rebound, eventually leading to the selective formation of γ-lactones.

Helper T cell immunity in humans with inherited CD4 deficiency.

CD4+ T cells are vital for host defense and immune regulation. However, the fundamental role of CD4 itself remains enigmatic. We report seven patients aged 5-61 years from five families of four ancestries with autosomal recessive CD4 deficiency and a range of infections, including recalcitrant warts and Whipple's disease. All patients are homozygous for rare deleterious CD4 variants impacting expression of the canonical CD4 isoform. A shorter expressed isoform that interacts with LCK, but not HLA class II, is affected by only one variant. All patients lack CD4+ T cells and have increased numbers of TCRαß+CD4-CD8- T cells, which phenotypically and transcriptionally resemble conventional Th cells. Finally, patient CD4-CD8- αß T cells exhibit intact responses to HLA class II-restricted antigens and promote B cell differentiation in vitro. Thus, compensatory development of Th cells enables patients with inherited CD4 deficiency to acquire effective cellular and humoral immunity against an unexpectedly large range of pathogens. Nevertheless, CD4 is indispensable for protective immunity against at least human papillomaviruses and Trophyrema whipplei.

Serum albumin prevents protein aggregation and amyloid formation and retains chaperone-like activity in the presence of physiological ligands.

Although serum albumin has an established function as a transport protein, evidence is emerging that serum albumin may also have a role as a molecular chaperone. Using established techniques to characterize chaperone interactions, this study demonstrates that bovine serum albumin: 1) preferentially binds stressed over unstressed client proteins; 2) forms stable, soluble, high molecular weight complexes with stressed client proteins; 3) reduces the aggregation of client proteins when it is present at physiological levels; and 4) inhibits amyloid formation by both WT and L55P transthyretin. Although the antiaggregatory effect of serum albumin is maintained in the presence of physiological levels of Ca(2+) and Cu(2+), the presence of free fatty acids significantly alters this activity: stabilizing serum albumin at normal levels but diminishing chaperone-like activity at high concentrations. Moreover, here it is shown that depletion of albumin from human plasma leads to a significant increase in aggregation under physiologically relevant heat and shear stresses. This study demonstrates that serum albumin possesses chaperone-like properties and that this activity is maintained under a number of physiologically relevant conditions.

Genome-wide patterns of intrahuman dengue virus diversity reveal associations with viral phylogenetic clade and interhost diversity.

Analogous to observations in RNA viruses such as human immunodeficiency virus, genetic variation associated with intrahost dengue virus (DENV) populations has been postulated to influence viral fitness and disease pathogenesis. Previous attempts to investigate intrahost genetic variation in DENV characterized only a few viral genes or a limited number of full-length genomes. We developed a whole-genome amplification approach coupled with deep sequencing to capture intrahost diversity across the entire coding region of DENV-2. Using this approach, we sequenced DENV-2 genomes from the serum of 22 Nicaraguan individuals with secondary DENV infection and captured ∼75% of the DENV genome in each sample (range, 40 to 98%). We identified and quantified variants using a highly sensitive and specific method and determined that the extent of diversity was considerably lower than previous estimates. Significant differences in intrahost diversity were detected between genes and also between antigenically distinct domains of the Envelope gene. Interestingly, a strong association was discerned between the extent of intrahost diversity in a few genes and viral clade identity. Additionally, the abundance of viral variants within a host, as well as the impact of viral mutations on amino acid encoding and predicted protein function, determined whether intrahost variants were observed at the interhost level in circulating Nicaraguan DENV-2 populations, strongly suggestive of purifying selection across transmission events. Our data illustrate the value of high-coverage genome-wide analysis of intrahost diversity for high-resolution mapping of the relationship between intrahost diversity and clinical, epidemiological, and virological parameters of viral infection.

Main mites associated with dermatopathies present in dogs and other members of the Canidae family.

Dermatological diseases of parasitic origin are one of the most frequent in the clinical practice of dogs and cats. Mites such as Sarcoptes scabiei, Otodectes cynotis, Demodex canis, and Cheyletiella spp., commonly affect domestic dogs. However, the impact generated by these mites on populations of wildlife animals and the mechanisms involved in their epidemiological dynamics are still not clear. In recent decades, the migration of populations and their interaction with domestic environments and vice versa have generated a worrying threat due to the transmission of some of these ectoparasites. Some reports have suggested that sarcoptic mange represents an emerging threat to wildlife. Given the outbreaks of greater magnitude and geographical extension. The objective of this review is to contribute to the state of the art of the main mites that cause dermatopathies in members of the Canis lupus familiaris family and other members of the Canidae family. For this, a systematic search was carried out in the Embase and PubMed databases. Infections caused by mites, mainly scabies, continue to be diseases with a worldwide distribution, affecting mammals and humans. Although they are long-standing diseases, the effects that are generated in wild canids are still unknown. A comprehensive evaluation is required to generate guidelines in favor of the conservation of some species of foxes and wolves present in different regions of the world.

Antimicrobial Resistance and Extended-Spectrum Beta-Lactamase Genes in Enterobacterales, Pseudomonas and Acinetobacter Isolates from the Uterus of Healthy Mares.

Antibiotic-resistant bacteria are a growing concern for human and animal health. The objective of this study was to determine the antimicrobial resistance and extended-spectrum beta-lactamase genes in Enterobacterales, Pseudomonas spp. and Acinetobacter spp. isolates from the uterus of healthy mares. For this purpose, 21 mares were swabbed for samples, which were later seeded on blood agar and MacConkey agar. The isolates were identified using MALDI-TOF and the antimicrobial susceptibility test was performed using the Kirby-Bauer technique. To characterize the resistance genes, a polymerase chain reaction (PCR) scheme was performed. Of the isolates identified as Gram-negative, 68.8% were Enterobacterales, represented by E. coli, Enterobacter cloacae, Citrobacter spp., and Klebsiella pneumoniae; 28.1% belonged to the genus Acinetobacter spp.; and 3.1% to Pseudomonas aeruginosa. A 9.3% of the isolates were multidrug-resistant (MDR), presenting resistance to antibiotics from three different classes, while 18.8% presented resistance to two or more classes of different antibiotics. The diversity of three genes that code for ESBL (blaTEM, blaCTX-M and blaSHV) was detected in 12.5% of the strains. The most frequent was blaSHV, while blaTEM and blaCTX-M were present in Citrobacter spp. and Klebsiella pneumoniae. These results are an alarm call for veterinarians and their environment and suggest taking measures to prevent the spread of these microorganisms.

iTRAQ-based proteomic profiling of breast cancer cell response to doxorubicin and TRAIL.

Breast cancer is a molecularly heterogeneous disease, and predicting response to chemotherapy remains a major clinical challenge. To minimize adverse side-effects or cumulative toxicity in patients unlikely to benefit from treatment, biomarkers indicating treatment efficacy are critically needed. iTRAQ labeling coupled with multidimensional LC-MS/MS of the enriched mitochondria and endoplasmic reticulum fraction, key organelles regulating apoptosis, has led to the discovery of several differentially abundant proteins in breast cancer cells treated with the chemotherapeutic agent doxorubicin followed by the death receptor ligand, TRAIL, among 571 and 801 unique proteins identified in ZR-75-1 and MDA-MB-231 breast cancer cell lines, respectively. The differentially abundant proteins represent diverse biological processes associated with cellular assembly and organization, molecular transport, oxidative stress, cell motility, cell death, and cancer. Despite many differences in molecular phenotype between the two breast cancer cell lines, a comparison of their subproteomes following drug treatment revealed three proteins displaying common regulation: PPIB, AHNAK, and SLC1A5. Changes in these proteins, detected by iTRAQ, were confirmed by immunofluorescence, visualized by confocal microscopy. These novel potential biomarkers may have clinical utility for assessing response to cancer treatment and may provide insight into new therapeutic targets for breast cancer.

Characterization of microbial communities and predicted metabolic pathways in the uterus of healthy mares.

Background: Culture-independent techniques have made it possible to expand the knowledge about the composition of bacterial communities present in the healthy uterus and their role in health and disease, mainly in humans. However, in animals like mares, there is a dearth of information regarding this area. Aim: To narrow this knowledge gap, the objective of this study was to identify and characterize the composition and function of the uterine microbiome of a group of Chilean purebred mares (CPM), an equine breed with the oldest genealogical record in South America and an economical important reproductive industry. Methods: From uterine biopsy samples obtained during estrus, DNA extraction and targeted sequencing were performed to investigate the bacterial diversity and its probable metabolic function. Results: CPM biopsy samples were characterized by having a varied microbial composition, where the four most relatively abundant phyla were Proteobacteria (69.6%), Firmicutes (21.1%), Bacteroidetes (7.8%), and Actinobacteria (1.06%); which made up 99.6% of the total identified phyla. In contrast, Actinobacteria and Fusobacteria were the phyla not identified in all samples. Of a total of 59 genera identified across all samples, Staphylococcus was the most abundant genus with an average relative abundance of 18.88%, followed by Pseudomonas (17.9%), Escherichia/Shigella (10.42%), and Klebsiella (9.92%). Conclusion: These findings contribute to the knowledge of microbes' presence in the uterus, while future studies are required to demonstrate the role of these microorganisms in health and disease.

Protein-PAINT: Superresolution microscopy with signaling proteins.

Superresolution techniques have advanced our understanding of complex cellular structures and processes but require the attachment of fluorophores to targets through tags or antibodies, which can be bulky and result in underlabeling. To overcome these limitations, we developed a technique to visualize the nanoscale binding locations of signaling proteins by taking advantage of their native interaction domains. Here, we demonstrated that pPAINT (protein point accumulation in nanoscale topography) is a new, single-molecule localization microscopy (SMLM) technique and used it to investigate T cell signaling by visualizing the Src homology 2 (SH2) domain, which is common in signaling molecules. When SH2 domain-containing proteins relocate to the plasma membrane, the domains selectively, transiently, and reversibly bind to preferred phosphorylated tyrosine residues on receptors. This transient binding yields the stochastic blinking events necessary for SMLM when observed with total internal reflection microscopy and enables quantification of binding coefficients in intact cells. We used pPAINT to reveal the binding sites of several T cell receptor-proximal signaling molecules, including Zap70, PI3K, Grb2, Syk, Eat2, and SHP2, and showed that the probes could be multiplexed. We showed that the binding half-life of the tandem SH2 domain of PI3K correlated with binding site cluster size at the immunological synapses of T cells, but that longer binding lifetimes were associated with smaller clusters for the monovalent SH2 domain of Eat2. These results demonstrate the potential of pPAINT for investigating phosphotyrosine-mediated signaling processes at the plasma membrane.