Diverse type I and type II methanotrophs cultivated from an Indian freshwater wetland habitat.

Wetlands are the main natural sources of methane emissions, which make up a significant portion of greenhouse gas emissions. Such wetland patches serve as rich habitats for aerobic methanotrophs. Limited knowledge of methanotrophs from tropical wetlands widens the scope of study from these habitats. In the present study, a freshwater wetland in a tropical region in India was sampled and serially diluted to obtain methanotrophs in culture. This was followed by the isolation of methanotrophs on agarose-containing plates, incubated under methane: air atmosphere. Methanotrophs are difficult to cultivate, and very few cultures of methanotrophs are available from tropical wetlands. Our current study reports the cultivation of a diverse community of methanotrophs from six genera, namely, Methylomonas, Methylococcus, Methylomagnum, Methylocucumis (type I methanotrophs) along with Methylocystis, Methylosinus (type II methanotrophs). A high abundance of methanotrophs (106-1010 methanotrophs/g fresh weight) was observed in the samples. A Methylococcus strain could represent a putative novel species that was also isolated. Cultures of Methylomagnum and Methylocucumis, two newly described type I methanotrophs exclusively found in rice fields, were obtained. A large number of Methylomonas koyamae strains were cultured. Our study is pioneering in the documentation of culturable methanotrophs from a typical tropical wetland patch. The isolated methanotrophs can act as models for studying methanotroph-based methane mitigation from wetland habitats and can be used for various mitigation and valorization applications.

Taurine dynamics in serum during the oestrous cycle in buffaloes.

Estrus identification is one of the common issues in buffaloes because of their short estrus duration and silent estrus problem. Hence, specific biomarkers facilitating in identifying the estrus stage would be helpful to buffalo farmers and researchers. In our previous studies, taurine, a non-protein amino acid that helps in the secretion of reproductive hormones such as GnRH, was found to be associated with postpartum anestrus in buffaloes. Therefore, the present study was conducted to explore the level of taurine in serum during different stages of the oestrous cycle in healthy cyclic buffaloes. Blood samples were collected from healthy cyclic buffaloes (n = 4), and taurine was estimated at the estrus (0th day), proestrus (-2nd day), metestrus (3rd day) and diestrus (+10th day) stages using TLC method. The days of the oestrous cycle were determined by ultrasonography and observation of behavioural signs by trained professionals. The results revealed that taurine was consistently present in the serum. However, the highest concentration of taurine was observed at the proestrus (0.20 ± 0.03 mg/mL) stage, which was greater (p < .05) than metestrus (0.10 ± 0.05 mg/mL) and diestrus (0.13 ± 0.03 mg/mL) stages, but comparable with the estrus stage. These results were also validated in the simulated population datasets of population size 6 to 10,000. Further, ROC curve analysis for the large simulated population indicated the efficiency of taurine to distinguish proestrus from metestrus and diestrus stages at a lower cutoff value of <0.1643 mg/mL with 60% sensitivity and specificity. Therefore, the present study concludes that serum taurine concentration could help in detecting proestrus stage of buffalo estrous cycle.

Quantification of cystine in human renal proximal tubule cells using liquid chromatography-tandem mass spectrometry.

Nephropathic cystinosis is characterized by abnormal intralysosomal accumulation of cystine throughout the body, causing irreversible damage to various organs, particularly the kidneys. Cysteamine, the currently available treatment, can reduce lysosomal cystine and postpone disease progression. However, cysteamine poses serious side effects and does not address all of the symptoms of cystinosis. To screen for new treatment options, a rapid and reliable high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed to quantify cystine in conditionally immortalized human proximal tubular epithelial cells (ciPTEC). The ciPTEC were treated with N-ethylmaleimide, lysed and deproteinized with 15% (w/v) sulfosalicylic acid. Subsequently, cystine was measured using deuterium-labeled cystine-D4, as the internal standard. The assay developed demonstrated linearity to at least 20 µmol/L with a good precision. Accuracies were between 97.3 and 102.9% for both cell extracts and whole cell samples. Cystine was sufficiently stable under all relevant analytical conditions. The assay was successfully applied to determine cystine levels in both healthy and cystinotic ciPTEC. Control cells showed clearly distinguishable cystine levels compared with cystinotic cells treated with or without cysteamine. The method developed provides a fast and reliable quantification of cystine, and is applicable to screen for potential drugs that could reverse cystinotic symptoms in human kidney cells.

Comprehensive proteomic analysis of buffalo milk extracellular vesicles.

Extracellular vesicles are secretory vesicles involved in cell-to-cell communication via their encapsulated cargo of proteins, lipids, and nucleic acids. Bovine milk provides a rich source of extracellular vesicles (mEVs) that have been studied as therapeutics and drug delivery systems. Therefore, insight into the mEV cargo, such as its proteome, may help in understanding the molecular mechanism underlying the potential health benefits attributed to the mEVs. Hence, mEVs were isolated from healthy buffalo milk after screening the milk somatic cell count. The total proteins of mEVs were analyzed using LC-MS, and 331 proteins were found commonly present among three buffalo milk samples. These proteins were primarily derived from extracellular regions and lysosomes. The major biological roles associated with the proteins were immune response, metabolism, and cell cycle regulation. The molecular functions of the proteins were transporter activity, catalytic activity, and GTPase activity. Further, comparative analysis with the previously available bovine mEVs proteome data showed 114 proteins to be newly identified in the buffalo mEVs. The biological pathways associated with these proteins may play a major role in muscle development. These findings shed a light on the potential health benefits of buffalo mEVs as therapeutics as well as drug delivery vehicles.

Purpose reflection benefits minoritized students' motivation and well-being in STEM.

Students from groups historically excluded from STEM face heightened challenges to thriving and advancing in STEM. Prompting students to reflect on these challenges in light of their purpose can yield benefits by helping students see how their STEM work connects to fundamental motives. We conducted a randomized, controlled trial to test potential benefits of reflecting on purpose-their "why" for pursuing their degrees. This multimethod study included 466 STEM students (232 women; 237 Black/Latinx/Native students). Participants wrote about their challenges in STEM, with half randomly assigned to consider these in light of their purpose. Purpose reflection fostered benefits to beliefs and attitudes about the major, authentic belonging, and stress appraisals. Effects were robust across race and gender identities or larger for minoritized students. Structural and cultural shifts to recognize students' purpose in STEM can provide a clearer pathway for students to advance.

Mucin and salt combination simulate typical fern-like pattern of buffalo saliva smear at estrus.

Estrus detection in buffaloes primarily relies on behavioral and physiological signs. Especially during summer, these signs are less prominent to recognize. Thus, estrus detection is a pronounced challenge within the realm of buffalo husbandry, particularly in the summer. Therefore, a simple and accurate estrus detection method is required for buffalo farmers. The observation of fern-like salivary crystallization patterns is one such simple method to detect estrus in buffaloes, bactrian camels, beagle bitches, and cows. However, the exact mechanism for the formation of typical fern-like is not known. We hypothesized that it might be because of the estrus-specific mucins and salts. To test this hypothesis, we prepared the smears by combining different concentrations of mucin type -2 (MUC2) and -3 (MUC3) with sodium chloride (NaCl). Microscopic examination confirmed that fern-like patterns resulted from a combination of the MUC3 and NaCl produced more realistic fern patterns than that of MUC2 or BSA with salt. To predict possible mucin and salt concentration showing natural fern-like patterns at the estrus stage in buffalo saliva, we constructed a guide tree of artificially generated fern-like patterns using an image analysis online tool. This computation analysis revealed that most of the natural buffalo estrus saliva samples showing typical fern-like patterns clustered in the cluster 2 of the guide tree comprising of 13 clusters. In the cluster 2, MUC3 in combination with the salt concentrations of 100, 150, and 250 mM was commonly found in a close proximity to the natural typical fern-like patterns of saliva smear of buffaloes at estrus. Conclusively, the buffalo saliva at estrus is predicted to have a gel-forming heavily glycosylated protein such as mucin along with at least 100 mM of NaCl. RESEARCH HIGHLIGHTS: Glycoprotein and salts combination replicates fern-like pattern of buffalo saliva at estrus. MUC3 and NaCl salt combination produces more realistic fern-like patterns compared with MUC2 or BSA and salt combination. MUC3 with NaCl at 100, 150, and 250 mM consistently resembled natural estrus saliva fern-like patterns. During estrus, buffalo saliva is expected to contain heavily glycosylated mucin and at least of 100 mM NaCl.

Recent advancements in targeted protein knockdown technologies-emerging paradigms for targeted therapy.

A generalized therapeutic strategy for various disease conditions, including cancer, is to deplete or inactivate harmful protein targets. Various forms of protein or gene silencing molecules, e.g., small molecule inhibitors, RNA interference (RNAi), and microRNAs (miRNAs) have been used against druggable targets. Over the past few years, targeted protein degradation (TPD) approaches have been developed for direct degradation of candidate proteins. Among the TPD approaches, proteolysis targeting chimeras (PROTACs) have emerged as one of the most promising approaches for the selective elimination of proteins via the ubiquitin-proteasome system. Other than PROTACs, TPD methods with potential therapeutic use include intrabody-mediated protein knockdown and tripartite motif-21 (TRIM-21) mediated TRIM-Away. In this review, protein knockdown approaches, their modes of action, and their advantages over conventional gene knockdown approaches are summarized. In cancers, disease-associated protein functions are often executed by specific post-translational modifications (PTMs). The role of TRIM-Away is highlighted in the direct knockdown of PTM forms of target proteins. Moreover, the application challenges and the prospective clinical use of TPD approaches in various diseases are also discussed.

In the Face of Opportunities: Facial Structures of Scientists Shape Expectations of STEM Environments.

Impressions of role leaders provide information about anticipated opportunities in a role, and these perceptions can influence attitudes about science, technology, engineering, and mathematics (STEM) pathways. Specifically, the facial structures of role leaders influenced perceived affordances of working with that person, such as the availability of communal and agentic opportunities (e.g., mentorship; achievement). STEM faculty with trustworthy (relative to dominant) faces were seen as valuing communal goals (Studies 1-3), and in turn, perceived as affording both communal and agentic opportunities in their research groups (Studies 2-3b). These heightened goal opportunities aligned with perceptions that trustworthy-faced advisors would enact more group-supportive behaviors (Study 2). Consequently, students anticipated fairer treatment and reported greater interest in labs directed by trustworthy- than dominant-faced leaders (Studies 3a-4a), even when images were accompanied by explicit information about leaders' collaborative behavior (Study 4b). The faces of leaders can thus function as the "face" of that role and the surrounding culture.

Detection and quantification of TIMP1 and miR-141 through RT-LAMP and TT-LAMP in serum samples during estrous cycle in buffalo.

Estrus identification is a common problem in the reproductive management of farm animals. Hence, several studies have been conducted to explore biomarkers for estrus detection. One of our previous studies identified the abundance of RNA biomarkers such as TIMP1 and miR-141 in buffalo saliva during the estrus stage. However, the level of these RNA biomarkers in buffalo serum during estrous cycle is undetected. Therefore, the present study was designed to quantify TIMP1 and miR-141 in serum during buffalo estrous cycle. Blood samples were collected in different stages of estrous cycle from four healthy cyclic buffaloes. The quantification of TIMP1 and miR-141 was performed with direct serum using RT-LAMP and TT-LAMP technologies, respectively. The LAMP amplification was confirmed by agarose gel electrophoresis and the color change was quantified in comparison to a non-template control using ImageJ software. A decreased abundance of TIMP1 at the diestrus stage and a decreasing trend of miR-141 from proestrus to diestrus stages were observed, which was further reinforced by simulated random populations generated with R programming. Specifically, TIMP1 was found significantly (P < 0.0001) abundant at estrus and metestrus stages as compared to the diestrus stage, whereas miR-141 was significantly (P < 0.001) higher during the proestrus stage as compared to the other stages of estrous cycle. The ROC curve analysis showed miR-141 to be a better biomarker than TIMP1 as it distinguished the proestrus stage from diestrus with a sensitivity and specificity of 83 % and 98 %. This study also marked the first use of TT-LAMP technology for rapid miRNA detection in livestock.

My Fair Lady? Inferring Organizational Trust From the Mere Presence of Women in Leadership Roles.

The history of male dominance in organizational hierarchy can leave a residue of mistrust in which women in particular do not expect fair treatment. The mere presence of a female leader relative to a male leader led perceivers to anticipate fairer treatment in that organization (Study 1) and greater projected salary and status (Study 2). This mere presence effect occurred uniquely through communal and not agentic affordances; these patterns emerged especially or only for women. Female leaders cued organizational trust in both male- and female-dominated industries (Study 3) and when they occupied different levels of the organizational hierarchy (Study 4). When information about organizational communal affordances is directly communicated, both female and male leaders signal trust (Study 5). The processes and practices of male-dominated organizational culture can leave a residue of mistrust, but viewing women in leadership is one beacon illuminating paths forward and upward.

Unpacking Motivational Culture: Diverging Emphasis on Communality and Agency Across STEM Domains.

The current research examined whether life sciences vs. engineering/physical sciences vary in the visibility and value of communality and agency. Overall, we find an emphasis on agency in engineering/physical sciences and a greater balance between communality and agency in the life sciences. We examine motivational culture as represented in environmental structures (Study 1), in signals sent and received in academic displays (Studies 2A-B), and in individual-level motives and cognitions (Studies 3-4). Study 1 analyzed archival course data to find that courses (N=11,222) in engineering/physical sciences included fewer collaborative assignments than courses in life sciences. Study 2A's content analysis documented that bulletin boards (N=68) in engineering/physical sciences academic buildings conveyed less communal purpose, and Study 2B found that participants (N=44) perceived greater communal purpose when viewing novel bulletin boards experimentally manipulated to include the cues identified in Study 2A. In Studies 3 (N=326) and 4 (N=110), engineering/physical science majors reported a strong agentic focus, compared to life science majors' more balanced focus. Further, the strong agentic focus of engineering/physical science students waned over time. This investigation of motivational cultures highlights the daily practices and institutional contexts that can shape individual-level motives and cognition related to engagement in STEM, both within and across different STEM pathways.

Direct knockdown of phospho-PTM targets mediated by TRIM21 can improve personalized treatment in breast cancer.

PURPOSE: In this work for the first time, we showed specific and direct knockdown of important oncogenic proteins of interest and their phospho-PTM targets in tripartite motif containing-21 (TRIM21) overexpressing breast cancer (BC) cells. We revealed the functional and therapeutic consequences of this protein knockdown approach called 'TRIM-ing'. METHODS: To target HER2, HER3, STAT3 or their activated forms, electroporation and puls-in transfection were standardized for mAb delivery in AU565 and MCF7 BC cell lines. Cancer cells were treated with HER2-targeted medicines (Trastuzumab and Neratinib) or STAT3 targeted inhibitors (Stattic and Niclosamide) with or without respective target TRIM-ing. Real-time PCR, immunoblotting, immunofluorescence, cytotoxicity, short- and long-term cell survival assessments were done following standard methodologies. 3-D structure modelling was used to verify the binding of mAb onto the STAT3 target. RESULTS: TRIM-ing of HER2 or HER3 receptors or their activated phospho-forms in BC cells showed rapid degradation of respective protein forms, shattering down the downstream signaling (p-ERK, p-AKT) that lasts for up to 7-8 days. This significantly inhibited BC survival (p < 0.001), showing a synergistic therapeutic effect with HER2 medicine trastuzumab or neratinib. Additionally, specific TRIM-ing ability of canonical pY705 or non-canonical pS727 PTMs of STAT3 protein was demonstrated in MCF7 cells, causing significant cytotoxicity (p < 0.05). TRIM-ing of STAT3 PTM, when combined with the same PTM-specific inhibitors, a synergistic treatment effect was observed. CONCLUSION: The work demonstrated that TRIM-ing could directly reduce various oncogenic targets or their specific activated form inside the cancer cells without compensatory pathway activation, a conundrum limiting the therapeutic benefit of current personalized medicines.

Out of the Lab and Into the World: Analyses of Social Roles and Gender in Profiles of Scientists in The New York Times and The Scientist.

Although representations of female scientists in the media have increased over time, stereotypical portrayals of science persist. In-depth, contemporary profiles of scientists' roles have an opportunity to reflect or to challenge stereotypes of science and of gender. We employed content and linguistic analyses to examine whether publicly available profiles of scientists from New York Times and The Scientist Magazine support or challenge pervasive beliefs about science. Consistent with broader stereotypes of STEM fields, these portrayals focused more on agency than communality. However, profiles also challenged stereotypes through integrating communality, purpose, and growth. This analysis also found similar presence of communal and agentic constructs for both female and male scientists. The current findings highlight the importance of considering counterstereotypic representations of science in the media: Communicating messages to the public that challenge existing beliefs about the culture of science may be one path toward disrupting stereotypes that dissuade talented individuals from choosing science pathways.

Putting Belonging in Context: Communal Affordances Signal Belonging in STEM.

A sense of belonging in a particular context is cued not only by the people in the role but by the affordances of the role-that is, the opportunities for goal pursuit. We investigate this role-based belonging in four studies documenting that the perceived affordances of social roles inform sense of belonging and convey known benefits of belonging. Perceiving more communal opportunities in naturalistic science, technology, engineering, and mathematic (STEM) settings was associated with heightened belonging in those roles (Studies 1-2). Experimentally manipulating collaborative activities in a science lab increased anticipated belonging in the lab and fostered interest, particularly among women (Study 3). Finally, mentally simulating communal affordances in a role promoted recovery from belonging threat: Considering communal opportunities in STEM facilitated recovery of STEM-specific belonging after recalling exclusion in STEM (Study 4). Investigations of role-based belonging offer the potential for both theoretical and practical advances.

Diverse type I and type II methanotrophs cultivated from an Indian freshwater wetland habitat

Wetlands are the main natural sources of methane emissions, which make up a significant portion of greenhouse gas emissions. Such wetland patches serve as rich habitats for aerobic methanotrophs. Limited knowledge of methanotrophs from tropical wetlands widens the scope of study from these habitats. In the present study, a freshwater wetland in a tropical region in India was sampled and serially diluted to obtain methanotrophs in culture. This was followed by the isolation of methanotrophs on agarose-containing plates, incubated under methane: air atmosphere. Methanotrophs are difficult to cultivate, and very few cultures of methanotrophs are available from tropical wetlands. Our current study reports the cultivation of a diverse community of methanotrophs from six genera, namely, Methylomonas, Methylococcus, Methylomagnum, Methylocucumis (type I methanotrophs) along with Methylocystis, Methylosinus (type II methanotrophs). A high abundance of methanotrophs (106–1010 methanotrophs/g fresh weight) was observed in the samples. A Methylococcus strain could represent a putative novel species that was also isolated. Cultures of Methylomagnum and Methylocucumis, two newly described type I methanotrophs exclusively found in rice fields, were obtained. A large number of Methylomonas koyamae strains were cultured. Our study is pioneering in the documentation of culturable methanotrophs from a typical tropical wetland patch. The isolated methanotrophs can act as models for studying methanotroph-based methane mitigation from wetland habitats and can be used for various mitigation and valorization applications. (AU)