bluuebunny/crossref_metadata_2025 huggingface dataset:
Teaching Essay Writing: When Good Intentions Lead to Bad Outcomes
Authors: Witchel Harry J.
Published: May 2022

This warning narrative explores the tension in student‐centered learning between attempting to teach individual students the skills of scientific writing in good faith versus encouraging student responsibility. There are often protocols to facilitate student learning that systematically apportion tasks between different members of staff. These protocols are reliable and well‐established, but in the face of a failing student, plainly some aspect of the protocol has gone wrong. Has the student fallen through a gap in the system, has a busy instructor given insufficiently personalized teaching, or has the student failed to take responsibility for their student‐centered learning? We conclude that deviating from protocols, even with due caution and substantive teaching experience, can still lead to conflicts.

In this narrative example, the author, a physiology educator, failed to follow protocol and ended up creating predictable problems for his colleagues. The author made this mistake despite the fact that he has also been awarded a National Teaching Fellowship (UK) and has also written a book “Writing for Biomedical Sciences Students” for Macmillan Study Skills. The tension between the student needing skills and the student needing to take responsibility highlights how learning in good faith (and teaching in good faith) is at the core of student‐centered learning. The case study looks at an anonymized group email trail among faculty to explore the sequelae of an optional “essay help session” for a group of students who have failed an essay assignment.

As the author ran an active learning workshop session for 8 students, a latecomer interrupted the proceedings to complain about her original mark in the form of not understanding what was wrong with the original failed essay. In our system, remarking is neither encouraged nor allowed except in extraordinary circumstances. The author, falling into the situation of wanting to teach in good faith, skimmed the essay during the class and found it on first glance to have only a series of subtle problems. The promise “to look into it after class” resulted in a series of group emails that suggested that the author ‐‐ despite extensive efforts to avoid the obvious conflict between faculty ‐‐ had undermined the original tutor while being absorbed by a student's aims that may have been in bad faith from the beginning. The student definitely took responsibility for addressing their failure, but not necessarily by learning the skills being taught. Despite the instructor's explicit recognition of the potential conflicts and acknowledgment of the important roles of other instructors, the conflicts can still arise.
Orlistat, an anti‐obesity remedy as a competitive lipase inhibitor, enhanced inflammatory reaction in the intestine
Authors: Katimbwa Dorsilla A.
Published: May 2022

Secondary gut metabolites derived from microbial fermentation of food in the gut have broad impacts on various aspects of host physiology. Our recent studies on obese mice treated with Orlistat, an anti‐obesity treatment, showed significantly altered gut microbial profile marked by an overabundance of Proteobacteria and possible alterations in secondary gut metabolites. Consequently, aqueous fecal extracts from treated and untreated mice groups were analyzed for anti‐oxidant and mitochondrial altering function using HCT‐116‐ARE and Caco2 cells under oxidative stress. The results of this study showed that fecal extracts in groups containing increased cecal concentraations of butyrate and propionate had reduced fold change in luciferase activity, thus, alleviating oxidative stress. Conversely, fecal extracts from high fat/orlistat treated mice resulted in a significant increase in fold change in the luciferase activity and maximal ATP production of cells under oxidative stress. Changes in secondary gut metabolites during obesity treatment may provide the necessary insight into the possible side effects seen during intervention.
Vitamin D Status and its Associations with Cytokines and Chemokines in Arab Adults Screened for COVID‐19
Authors: Al‐Daghri Nasser , Amer Osama , Aljohani Naji , Alfawaz Hanan , Hussain Syed Danish , Alnaami Abdullah , Sabico Shaun
Published: May 2022

Observational studies have overwhelmingly demonstrated that vitamin D deficiency is a risk factor for coronavirus disease‐19 (COVID‐19) infection and severity. However, serum 25(OH)D may act as a negative acute phase reactant and therefore an unreliable marker for vitamin D status post‐inflammatory insult. This study evaluated the serum levels of 25(OH)D, 34 cytokines and chemokines in 220 participants (82 control and 138 SARS‐CoV‐2 patients). Serum 25(OH)D levels were significantly lower in the SARS‐CoV‐2 group than controls. Serum IP‐10, MCP‐1, CRP, IFNγ, IL‐10, IL‐13, IL‐17α, IL‐23, and IL‐6 were significantly higher in COVID‐19 patients compared to healthy control. Results showed that serum levels of VEGF, IFNγ, IL‐13, and IL‐5 were significantly higher in male patients compared to females. 25(OH)D was significantly correlated with EFG (R=0.39, p<0.05) and IL‐15 (R=0.39, p<0.05) in male patients, while inversely correlated with CRP (R=‐0.51, p<0.05) in female patients. In conclusion, we recommend 25(OH)D supplementation among high‐risk individuals and SARS‐CoV‐2‐infected individuals. Additionally, the upregulated cytokines might serve as therapeutic targets to modulate the heightened inflammation and disease severity, moreover, they could be helpful in the early screening of critical illness, diagnosis and treatment of SARS‐CoV‐2.
Novel Anosmia‐Inducing Compounds for Environmentally Friendly Mosquito Vector Control: Structural Determinants of ORco Ligands Antagonizing Odorant Receptor Function
Authors: Iatrou Kostas , Kythreoti Georgia , Thireou Trias , Karoussiotis Christos , Georgoussi Zafiroula , Zographos Spyros E. , Liggri Panagiota , Michaelakis Antonios , Schulz Stefan
Published: May 2022

Insect repellents are important means of personal protection against bites from mosquitoes carrying various pathogens. Odorant‐dependent behaviours are triggered by odor ligands bound to odorant receptors. Insect odorant receptors are heterotetrameric ligand‐gated cation channels composed of an obligatory subunit, ORco, and one of many variable subunits, ORx, to which odorant ligands bind. Upon expression ex vivo, ORco forms homomeric channels gated by specific ligands acting as channel agonists. In past studies, we identified ligands of natural origin that bind to the African malaria mosquito vector Anopheles gambiae ORco (AgamORco). Some of these ligands function as specific ORco channel antagonists, orthosteric or allosteric relative to a predicted ORco agonist binding site, and cause severe inhibition of the olfactory function in a variety of mosquito species. Given that ORco is evolutionarily conserved, we hypothesized that it should be feasible to develop an AgamORco‐based screening platform allowing fast identification of new ORco ligands acting as mosquito odorant receptor antagonists causing anosmia‐like deficits to mosquitoes.

We are now reporting on the compilation of common structural features of previously identified orthosteric AgamORco antagonists and generation of a ligand‐based pharmacophore. In silico screening of a collection of volatile compounds of natural origin resulted in identification of several AgamORco ligand hits. Parallel cell‐based screening of the same compounds against AgamORco was also employed in order to identify ligands acting as AgamORco antagonists. The screening resulted in identification of several ligands inhibiting AgamORco channel function ex vivo by at least 40% and inducing anosmic behaviors to Aedes albopictus mosquitoes in vivo. To test the pharmacophore model for binding site specificity, binding competition assays were undertaken, which distinguished the identified antagonists into orthosteric and allosteric ones. Direct comparisons of the pharmacophore predictions with the results of the cell‐based screen revealed that the pharmacophore predicted correctly 100% of the orthosteric antagonists and none of the allosteric ones. These results confirm the pharmacophore's value for in silico prediction of AgamORco orthosteric ligands. Upon combination with the AgamORco‐specific cell‐based screening platform, the pharmacophore provides a valuable tool for fast identification of multiple, ecologically friendly vector control agents. Finally, to confirm the predicted AgamORco agonist binding site and determine the binding orientation of orthosteric antagonists in it, we are assessing the interactions of these ligands with specific amino acids in the AgamORco binding pocket by site‐specific mutagenesis.
ThrRS is a novel regulator of STAT3 activation
Authors: Dai Chong , Chen Jie
Published: May 2022

Signal transducer and activator of transcription 3 (STAT3) plays crucial roles in multiple biological processes, such as cell proliferation, migration, and inflammation. Elevated STAT3 activity is found in many types of cancer. In a screen for non‐translational targets of threonyl‐tRNA synthetase (ThrRS), we found in HEK293 cells STAT3 phosphorylation at Y705, both at steady‐state and stimulated by IL‐6, to be reduced by ThrRS knockdown. Conversely, overexpression of ThrRS enhanced STAT3 phosphorylation. Consistently, nuclear translocation and transcriptional activity of STAT3 were also inhibited by ThrRS knockdown and activated by ThrRS overexpression. This function of ThrRS was independent of its canonical activity of aminoacylation. In searching for a molecular mechanism of this novel regulation, we found that ThrRS physically interacted with both STAT3 and JAK1/2, and that knockdown of ThrRS dampened the interaction between STAT3 and JAK1/2. Furthermore, STAT3 and JAK1/2 interacted with the aminoacylation domain and the editing domain of ThrRS, respectively. Taken together, our observations suggest that ThrRS may serve to recruit STAT3 to JAK for activation. Finally, elevated ThrRS expression was found to accompany hyperactivity of STAT3 in breast cancer cells, and knockdown of ThrRS reduced STAT3 phosphorylation and suppressed cancer cell proliferation. In conclusion, we have identified ThrRS as a non‐canonical regulator of STAT3 activation and a potential anti‐cancer therapeutic target.
Investigation into the Structure and Activity of the Arabidopsis β‐amylase BAM1
Authors: Riney McKayla B. , Monroe Jonathan , Berndsen Christopher
Published: May 2022

Starch in plants is a large polymer of glucose that stores excess glucose for times of limited photosynthesis. Starch is used in many industrial processes such as laundry and papermaking. In plants, regulation of starch storage and degradation is a multifaceted system that allows the plant to adjust carbohydrate storage in response to a variety of environmental and developmental conditions. However, we are unsure how this process is regulated. β‐amylases (BAMs) hydrolyze starch to produce maltose. Plants contain many BAM proteins and possibly each has a unique function and regulatory mechanism, although this is an area of active research. The degradation of starch by BAM1 is activated when the plant undergoes osmotic stress. BAM1 is suggested to be regulated by a redox reaction, however, the precise regulatory mechanism is unclear although it is thought to involve disulfide formation between an N‐terminal cysteine and another within the amylase domain. A better understanding of the structure and the activity of BAM1 will allow us to improve many crucial industries. To address the structure of BAM1, we purified the protein from E. coli cells and ensured that the protein was folded using circular dichroism. We then collected small‐angle X‐ray scattering (SAXS) data on BAM1 and analyzed the data to determine solution dimensions of BAM1. Those data were then compared against SAXS data of other BAM proteins collected in our lab. The preliminary SAXS data and comparison support BAM1 being a monomeric protein with an extended N‐terminus. Another part of this project was to determine the activity of BAM1 and whether the activity is affected by oxidation. We are optimizing the Bicinchoninic acid (BCA) assay, as well as a maltose‐binding protein green fluorescent protein (MBP‐GFP) assay to investigate the activity of BAM1 and the effects of oxidizing conditions. Further investigation into the disulfide bond will be done using DSF, and CD.
Structural basis for specific activation of the Rab11 GTPase by the TRAPPII complex
Authors: Bagde Saket R. , Fromme J. Christopher
Published: May 2022

Secretion is an essential cellular function in which proteins and lipids are transported from the Golgi to the plasma membrane via secretory vesicles. The formation of secretory vesicles is regulated by activation of the Rab GTPase Rab11. The activation of Rab GTPases at membrane compartments is dependent on the localization of their specific Guanosine Exchange Factors (GEFs), which catalyze the exchange of GDP with GTP and stabilize the active GTPase on the membrane. The multisubunit TRAPPII complex acts as Rab11 GEF and recruits it to the trans‐Golgi network (TGN) membrane. More than 60 different Rabs are present in mammals and budding yeast has 11 different Rabs. Specific localization of each of these Rabs to different membrane compartments is important for determination of organelle identity and membrane organization of these compartments. While multiple GTPases and GEFs localize at the TGN, the mechanistic details of how TRAPPII specifically activates Rab11, and not other GTPases, is unknown. Moreover, another related GEF, the TRAPPIII complex, shares the same catalytic subunits with TRAPPII and yet activates another GTPase, Rab1. A steric gating mechanism has been proposed in which TRAPPII selects against Rab1 based on the length of the C‐terminal hypervariable domain. We determined the structure of yeast TRAPPII bound to its substrate Rab11 using cryo‐EM at an overall resolution of 3.7Å. Analysis of the atomic structure has revealed specific interactions between Rab11 and the TRAPPII complex, and how TRAPPII interacts with the membrane surface. We tested the physiological relevance of these observed interactions using in vivo functional studies and in vitro reconstitution of nucleotide exchange. We show that the TRAPPII subunit Trs130 provides a ‘leg’ which lifts the TRAPP catalytic site above the membrane, thereby preventing access to the catalytic site by Rab1. TRAPPIII complex selects against Rab11 based on repulsive interactions with the catalytic subunits. We also show an alternative conformation of the TRAPPII complex which may facilitate access of Rab11 to the TRAPP catalytic site. Taken together, these experiments reveal the mechanism of specific activation of Rab11 on the membrane by TRAPPII, a key step for initiation of secretory vesicle formation. As Rab11 and the TRAPPII complex are conserved throughout eukaryotes, this mechanism may be widely conserved across all eukaryotic secretory systems.
Differential remodelling of visceral and pericardial adipose tissue in response to stress and exercise
Authors: Boateng Samuel , Ige Susan , Dallas Mark , Chantler Paul
Published: May 2022

Obesity is driven by an imbalance between caloric intake and energy expenditure, causing excessive storage of nutrients in adipose tissue at different sites around the body. Increased visceral adipose tissue (VAT) is associated with diabetes, whilst pericardial adipose tissue (PAT) is associated with cardiac pathology. Adipose tissue can expand either through cellular hypertrophy or hyperplasia, with the former correlating with decreased metabolic health in obesity. The aim of this study was to determine how VAT and PAT remodel in response to obesity, stress and exercise.

Here we have used the obese Zucker rats which develop hyperphagia with reduced energy expenditure, resulting in morbid obesity as a result of leptin resistance. At 9 weeks of age lean (LZR) and obese (OZR) Zucker male rats were treated with or without unpredictable chronic mild stress or exercise for 8 weeks. To determine the phenotype for PAT and VAT, tissue cellularity and gene expression were analyzed. Tissue cellularity was determined following haematoxylin and eosin staining, whilst qPCR was used to examine gene expression.

PAT adipocytes were significantly smaller than those from VAT and had a more biege‐like appearance. In the OZR group, VAT adipocyte cell size increased significantly compared with LZR, whilst PAT showed no change. Exercise and stress resulted in a significant reduction in VAT cellularity in OZR, whilst PAT showed no change. This suggests that PAT cellularity does not remodel significantly compared with VAT. Picro sirus red staining indicated that PAT had significantly more fibrosis than VAT. However, this fibrosis was decreased following stress treatment. This data indicate that the extracellular matrix of PAT is able to remodel more readily than in VAT.

Gene expression analysis showed that PAT expressed higher levels of UCP1 compared with VAT, indicating that it has a more brown‐like phenotype. In the LZR group, exercise increased insulin receptor substrate 1 (IRS1) in PAT, but was decreased in the OZR group. On the other hand, in VAT, exercise decreased IRS1 in LZR, whilst increasing it in the OZR. This suggests that in obesity, VAT is more responsive to exercise and subsequently becomes less insulin resistant compared with PAT. In VAT, stress increased PPAR‐γ expression in the OZR group but decreased it in the PAT. This suggests that in obesity, stress increases adipogenesis more significantly in the VAT compared with PAT.

Taken together, our data suggest that VAT is considerably more plastic than PAT and remodels rapidly in response to obesity, exercise and psychological stress.
Secreted Proteins, Lipids and Low‐Molecular‐Weight Metabolites as Early Biomarkers of Human Proximal Tubular Cell Exposure to Nephrotoxic Agents
Authors: Lash Lawrence H. , Mathieu Patricia , Stemmer Paul M. , Rosati Rita , Hu Xin , Jones Dean P.
Published: May 2022

Mitochondria and other organelles are early and sensitive targets for numerous nephrotoxic drugs and environmental contaminants. To prevent irreversible kidney injury from such chemical exposures it is critical to identify indicators (biomarkers) that correspond with the exposures before significant injury occurs. Primary cultures of human proximal tubular (hPT) cells were used as the model system to test the hypothesis that exposure to low, physiologically relevant concentrations of these agents causes release of proteins, lipids and metabolites into the extracellular space that correlate with specific exposures and cytotoxicity. Previous work with the nephrotoxic and mitochondrial‐targeting toxicant S‐(1,2‐dichlorovinyl)‐L‐cysteine (DCVC) confirmed that a large proportion (43%) of proteins recovered in the extracellular medium was of mitochondrial origin. Three of the most abundant proteins detected, for which levels were increased by 5‐ to 10‐fold by DCVC, were sulfite oxidase (mitochondrial), keratins (cytoskeletal), and HSP90 (cytoplasm). Comparisons of proteins detected in media from cells exposed to cisplatin (CDDP) vs. Control or polymyxin B (PmxB) vs. Control showed several commonly increased proteins. As with results from DCVC exposure, keratins and HSP90 were detected. The only mitochondrial protein that increased in abundance in supernatants from toxicant‐treated cells was carbamoyl‐phosphate synthase (1.4‐ and 2.3‐fold higher in supernatants from CDDP‐ and PmxB‐treated cells, respectively). Metabolomic analysis showed moderate changes (429 features at raw P < 0.05) for CDDP‐treated cells vs. Control and moderate to high changes (619 features at raw P < 0.05) for PmxB‐treated cells vs. Control. For CDDP. There was consistently lower release of 2‐aminoadipate 6‐semialdehyde and differences in purines, lipid species, and several amino acids. For PmxB, there was consistently lower release of aminoadipic acid and 2‐aminoadipate 6‐semialdehyde and differences in release of lipid species and lower release of phosphoenolpyruvate. These findings support the overall hypothesis and identify multiple candidate biomarkers of early exposure to diverse nephrotoxicants.
Mitochondrial Calcium Transporters Regulate Autophagy
Authors: de Miranda Ramos Vitor , Cabral‐Costa João Victor , Serna Julian D. , da Cunha Fernanda M. , Kowaltowski Alicia J.
Published: May 2022

Autophagy is an essential cellular process regulated by intracellular calcium signals, which play an important role in autophagic activation during metabolic changes. Calcium transporters are present in mitochondria, an organelle able to uptake and release calcium ions, thus participating in cellular calcium signaling. Uptake by mitochondria is mediated by the mitochondrial calcium uniporter (MCU), while extrusion occurs through the mitochondrial sodium/lithium/calcium exchanger (NCLX). The aim of this work was to investigate how MCU and NCLX affect autophagy. To do so, we evaluated makers of autophagic activity in murine Aml‐12 hepatic cells transfected with siRNAs targeting either MCU or NCLX expression, leading to genetic knockdown (KD). Additionally, we investigated the autophagic response to serum/amino acid starvation and rapamycin (mTORC1 inhibitor) treatment in Aml‐12 cells with NCLX KD or pharmacological inhibition by CGP37157 (CGP). Using a cell line stably expressing the autophagic probe LC3‐GFP‐mCherry, we observed that NCLX KD leads to impaired autophagosome formation under basal conditions. Curiously, this effect was associated with a significant decrease in mRNA expression of LC3A and LC3B genes, while the expression of other autophagy‐related genes, such as TFEB, ATG5, ATG12, and ATG7, was upregulated or unchanged. Conversely, MCU KD led to an apparent increase in autophagosome and autolysosome numbers, indicating enhanced autophagic activity. Interestingly, MCU KD also led to decreased expression of LC3A, but not LC3B. The expression of TFEB, ATG12, ATG5, and ATG7 were decreased or unchanged by MCU KD. After autophagic stimulation by serum/amino acid starvation, the levels of LC3 II were lower in NCLX KD cells compared to negative control in the presence or absence of bafilomycin A1, which indicates a reduction of autophagic flux. The levels of LC3 I were significantly lower in NCLX KD cells under basal and stimulated conditions, corroborating the decreased LC3 mRNA levels observed. Importantly, these effects were also observed using CGP to inhibit NCLX. The reduction of autophagic flux by NCLX KD or CGP was not observed in cells treated with rapamycin. We also measured the levels of phosphorylated 4E‐BP1 as an indication of mTORC1 activity. As expected, serum/amino acid starvation and rapamycin decreased the levels of p‐4E‐BP1; however, this decrease was modulated by CGP only in starved cells, indicating that NCLX may affect mTORC1 activity in an upstream pathway. In conclusion, we show that mitochondrial calcium transporters are novel autophagy‐regulating pathways: MCU modulates autophagic activation under basal conditions, while NCLX maintains autophagic activity under basal and stimulated conditions.
Breast Cancer Revisited: Stress Induced Conspiracy, Sensitivity and Specificity
Authors: Malik Samina , Waquar Sulayman , Malik Arif
Published: May 2022

INTRODUCTION

Cancer and related disorders remain as one of the most devastating ailments worldwide. It signifies the importance to devise particular techniques, which could help in attenuating the effects of breast cancer and related outcomes. Primary aim of the current study is to revisit the role of inflammatory and stress markers in the development and progression of breast cancer and to determine their sensitivity and specificity.

MATERIAL AND METHODS

A local sample of premenopausal Pakistani women (age 40 ± 3.6) diagnosed with breast cancer (n = 498) at Jinnah Hospital Lahore, and equal number of controls (age 36 ± 5.2) were selected for the current case control study after informed consent. Serum samples were separated and estimated for MMPs, ILs, HSPs, TNF‐α, INOS and Vitamin D on ELISA, whereas, MDA, SOD, NO, CAT, GSH, Vitamin A and D were estimated on Spectrophotometry.

RESULTS

Levels of MDA, ILs, MMPs, TNF‐α, NO, INOS and HSPs were significantly higher in the breast cancer patients, but the levels of SOD, CAT, GSH, Vitamins A, C and D were significantly lower, determining the relatively positive relationship of oxidants and negative relationship of antioxidants with the increased incidence of pathology among the patients. Furthermore, HSP‐27, MMP‐9 and IL‐1 levels revealed remarkable sensitivity and specificity on ROC analysis.

CONCLUSION

It concludes that HSP‐27, MMP‐9 and IL‐1 may serve as diagnostic and prognostic markers. Furthermore, it can be extrapolated how the increased levels of oxidative and nitrosative stress species along with inflammatory cytokines predispose to the incidence of cell invasion, cell migration and epithelial‐mesenchymal transition. Therefore, it infers that increase in growth factors i.e., VEGF, TGF‐β and Bcl2 under the influence of said variables play a crucial role in the metastasis of breast cancer.
Mutational analysis of the oligomerization domain of the HERV‐K Rec protein
Authors: Baier Katherine , Morell Michael , O'Carroll Ina P.
Published: May 2022

Retroviruses insert their genetic material into the host’s genome. When a retrovirus infects a germ cell, its sequence is passed down to subsequent generations‐‐it becomes endogenized. These human endogenous retroviruses (HERVs) remain mostly dormant and are activated in the presence of external signals (e.g. irradiation) or internal signals. Expression of HERV‐K, the most active human endogenous retrovirus, has been associated with cancer and neurological disorders, such as ALS. They also appear to regulate the innate immune system. For one subset of HERV‐K, nuclear export of the RNA genome relies on the successful interaction between a sequence on the viral genome, RcRE, and a viral protein, Rec. The Rec protein is produced from fully‐spliced mRNA and then imported back into the nucleus to bind and oligomerize on the RcRE region of the unspliced viral mRNA. Rec oligomerization is an essential feature of its function and, thus, a potential target for therapeutic intervention. To understand the mechanism of Rec‐RcRE complex formation, Rec variants were constructed via PCR mutagenesis. Residues of segments previously shown to be involved in multimerization were individually substituted by alanine. Functional assays using a RcRE reporter plasmid will be performed on HEK‐293 cells to assess the involvement of each Rec residue in oligomerization.
Mechanism and Evolution of [4+2] Cyclases in Monoterpene Indole Alkaloid Biosynthesis
Authors: DeMars Matthew D. , Nakamura Yoko , Kamileen Mohamed O. , Sanders Jacob N. , Paetz Christian , Houk K. N. , O'Connor Sarah E.
Published: May 2022

Monoterpene indole alkaloids (MIAs) comprise a large and diverse class of plant natural products, many of which exhibit potent biological activities such as anticancer, antimalarial, and antihypertensive. While the biosynthetic construction of these compounds has been a subject of intensive study for the past six decades, the identification of specific pathway genes remains an active area of investigation. MIAs belonging to the aspidosperma and iboga structural classes are produced only by select plants in the Apocynaceae (dogbane) family. The most well‐known of these is Catharanthus roseus, which produces the bisindole anticancer compounds vinblastine and vincristine through a complex pathway involving more than thirty individual enzymatic steps. Recently, we characterized three carboxylesterase‐like enzymes from C. roseus and Tabernanthe iboga that catalyze regio‐ and enantiodivergent [4+2] cycloadditions to generate the aspidosperma (tabersonine synthase, TS) and iboga (catharanthine synthase, CS; coronaridine synthase, CorS) scaffolds from a common biosynthetic intermediate. While crystal structures, computational docking, and site‐directed mutagenesis provided some key insights into the catalytic mechanism and cyclization specificity of these enzymes, several important questions remained unanswered. Here, we use a combination of computation and experiment to demonstrate that the [4+2] cyclases in MIA biosynthesis employ a stepwise rather than a concerted mechanism of cyclization. We also isolate and characterize the direct product of the CorS reaction and show that it can serve as a branch point in the biosynthesis of both iboga and pseudo‐aspidosperma alkaloids. Using a phylogenetic approach, we subsequently explore the evolution and diversification of MIA cyclase function. Reconstruction and functional analysis of ancestral enzymes allows us to trace an evolutionary trajectory from carboxylesterase to TS, from TS to CorS, and from CorS to CS. We further generate and test a panel of mutants based on both extant and ancestral cyclases to probe the molecular basis for differential cyclization activity. Our results provide insight into the mechanism and evolution of a unique class of biosynthetic enzymes and offer a means by which they could be leveraged for the facile production of new MIAs.
Vascular and glomerular vs. proximal tubular components in calcineurin inhibitor‐related nephrotoxicity
Authors: Popovic Suncica , Demirci Hasan , Yilmaz Duygu E. , Dittmayer Carsten , Ellison David H. , Mutig Kerim , Bachmann Sebastian
Published: May 2022

Calcineurin inhibitors (CNI) are widely in use for immunosuppression in transplant recipients, but their nephrotoxicity may cause or aggravate renal disease. CNI pathology includes vascular and tubulointerstitial alterations.

We have compared the effects of cyclosporine A (CsA) and tacrolimus (Tac) upon chronic administration in a rat model. We have tested whether Tac caused less damage than CsA. To this end, functional as well as glomerular and tubulointerstitial parameters were compared.

Adult Wistar rats received CsA (25 mg/kg/d), Tac (2 mg/kg/d), or vehicle via subcutaneously implanted minipumps. After four weeks, rats were sacrificed and organs removed for protein analysis or perfusion‐fixed and kidneys analyzed for histopathology. NRK cells were treated with graded concentrations of CNI for 24h. For evaluation, confocal immunohistochemistry, EM nanotomy, EM immunostaining, Western blot, and clinical chemistry methods were applied.

In rats, CsA and Tac produced distinct alterations in the tubulointerstitium. With CsA, dysmorphic lysosomes with peripheral LAMP1 signal, autophagic and mitophagic vacuoles caused epithelial decay of the initial proximal tubule, while glomerular changes were less pronounced. With Tac, glomerulosclerosis and damage encroachment to proximal tubule were more prominent. Both drugs induced fibrotic upregulation of alpha‐smooth muscle actin in the interstitium. Alterations in unfolded protein response (UPR) parameters included rises in p‐eIF2α, p‐PERK, p‐IRE1, sXBP1, CHOP in CsA‐, but not in Tac‐treated rats. Epithelial TUNEL signal was stronger in the CsA than in the Tac groups. Results were verified in NRK cells using graded caspase‐3 and lysosome formation criteria.

Data revealed distinct nephrotoxicity patterns in the two examined immunosuppressive medications. While CsA seems to be primarily tubulotoxic, a stronger glomerular component was detected with Tac. Results may serve to better adjust treatment combinations in transplant patients.
Understanding The Motives Of Body Donors
Authors: Smith Claire , Munro Ross , Davies Ceri , Wilkinson Tracey , Shaw Hannah , Claridge Kim , Llewellyn Sarah , Mc Ateer Philomena , Ward Siobhan , Farsides Tom
Published: May 2022

In the United Kingdom (UK) and Ireland approximately 1,400 body donors for Anatomical Examination are needed per year for the education and training of medical and allied health care professionals. The current study aimed to explore prospective donors’ motives, beliefs and desires about body donation to medical schools in England, Ireland, Scotland and Wales, with the hypothesis that their attitudes had not changed since the last such study in the UK in 1995 and that their motivation remained largely altruistic. A prospective, questionnaire‐based study of 821 potential donors over a 12 month period revealed a high level of agreement in answers to a number of questions exploring their motivation. Ninety six % of donors agreed they wished to improve the education of health care professionals, 92% agreed it was to help improve health care for patients, 78% considered that it provided a ‘good ending’ to their life. However, answers to two questions demonstrated a split viewpoint. When asked if saving relatives money was a motive 43% disagreed and 32% agreed, which was a marked increase compared to the 1995 study. A similar trend was seen when it came to sparing relatives inconvenience, with 46% of potential donors answering that this was not a motivating factor, but an increased 29% agreeing that it was. Inconvenience may mean different things to different people; practical, financial and emotional. Donors also reported differing views on avoiding traditional burial rituals with 46% agreeing that body donation helped them avoid such rituals, reflecting that some donor attitudes have changed since the last survey. Understanding donors’ altruistic motives is important, but so is recognizing some of the practical considerations important to them and how these may change with time. Therefore, the results of the current study help further our understanding of how we can support socially acceptable and ethical donation practices, including improved resources for donors, as well as also match future needs of the sector.
Pulmonary Endothelial Tau Aggregation After Infection
Authors: Lin Mike T. , Choi Chung‐Sik , Gwin Meredith , Voth Sarah , Kolb Claire , Zhou Chun , Nelson Amy R. , deWeever Althea , Koloteva Anna , Annamdevula Naga S. , Murphy James M. , Wagener Brant M. , Pittet Jean‐Francois , Lim Ssang‐Taek S. , Balczon Ron T. , Stevens Troy T.
Published: May 2022

Patients who recover from nosocomial pneumonia oftentimes exhibit long‐lasting cognitive impairment comparable to what is observed in Alzheimer’s Disease patients. We previously hypothesized that the lung endothelium contributes to infection‐related neurocognitive dysfunction, since bacteria‐exposed endothelial cells release a form(s) of cytotoxic tau that is sufficient to impair long‐term potentiation in the hippocampus. However, lung endothelial tau isoform(s) have yet to be resolved and it remains unclear whether the infection‐induced endothelial cytotoxic tau can trigger neuronal tau aggregation which is the major hallmark of several neuropathologies. Here, we demonstrate that lung endothelial cells express a big tau isoform and three additional tau isoforms that are similar to neuronal tau, each containing four microtubule‐binding repeat domains, and that tau is expressed in lung capillaries in vivo. To test whether infection elicits endothelial tau capable of causing transmissible tau aggregation, cells were infected with P. aeruginosa. The infection‐induced tau released from endothelium into the medium induced neuronal tau aggregation in reporter cells, including reporter cells that express either the four microtubule‐binding repeat domains or the full‐length tau. Infection‐induced release of pathological tau variant(s) from endothelium, and the ability of the endothelial‐derived tau to cause neuronal tau aggregation, was abolished using tau knockout cells. After bacterial lung infection, brain homogenates from wild‐type mice, but not from tau knockout mice, initiated tau aggregation. Notably, plasma samples obtained from pneumonia‐positive patients showed significantly higher tau aggregation activity when compared to pneumonia‐negative plasma samples. Thus, bacterial pneumonia initiates the release of lung endothelial‐derived cytotoxic tau into the circulation, which is capable of propagating a neuronal tauopathy.
Evidence of Sex Differences in the Endothelin Pathway in Diabetes
Authors: Derella Cassandra C. , Thomas Jeffrey , Layman Lawrence C. , Harris Ryan A.
Published: May 2022

Background

The endothelin system has been identified as a key regulator of vascular function and is comprised of two receptors; a constricting A‐receptor (ETAR) and dilating B‐receptor (ETBR). Sex differences in the ET‐1 pathway have been established. Specifically, when compared to men, activation of the ETBR in women results in a favorable dilatory response. In the presence of diabetes; however, the endothelin system in general, is negatively altered. Whether sex differences in the endothelin pathway are observed in type 1 diabetes (T1D), is unknown. Thus, the present investigation sought to test the hypothesis that ETB receptor function is reduced in women with T1D compared to men with T1D.

Methods

Premenopausal women (n=2, HbA1c=10.4±0.8%, age=29±13 years, BMI=29.9±2.8 kg/m2) and men (n=4, HbA1c=8.5±1.0%, age=22±3 years, BMI=21.5±2.8 kg/m2) with a clinical diagnosis of T1D were recruited to participate. Three microdialysis fibers were inserted into the dermal layer of the right forearm where cutaneous blood flow was measured using laser speckle contrast imaging. Microvascular function was determined by a ten dose incremental perfusion of acetylcholine (Ach; 10‐2‐2*103 M). Endothelin receptor function was determined two ways: 1) by a seven dose incremental perfusion of ET‐1 (10‐20‐10‐8 M), and 2) by a seven dose incremental perfusion of ET‐1 (10‐20‐10‐8 M) combined with the selective ETAR antagonist, BQ‐123 (750 nM). The dose response area under the curve (AUC) for cutaneous vascular conductance of each fiber was calculated for each individual.

Results

Women with T1D had a significantly greater BMI (p=0.026) and HbA1C(p=0.089) compared to men with T1D. The Ach dose response AUC was greater (p=0.135) in women with T1D (753.5±12.7 AU) when compared to men with T1D (691.7±61.1 AU). The ET‐1 + BQ‐123 dose response AUC tended (p=0.298) to be lower in women with T1D (295.5±14.1 AU) when compared to men with T1D (357.8±98.6 AU). A similar lower response in women was observed for the ET‐1 AUC (p=0.102; men: 1411.9±508.9 AU vs women: 821.7±38.5 AU).

Conclusion

These data suggest that, compared to men with T1D, microvascular function is greater in women, albeit weighing more and having a higher HbA1c. In contrast, preliminary findings indicate that ETBR function may be impaired in women with T1D compared to men with T1D.
MK2 Phosphorylates Caspase‐3, Facilitates Nuclear Translocation of Caspase 3, and Regulates Apoptosis
Authors: Del Rosario Othello Lance , Suresh Karthik , Shimoda Larissa , Rane Madhavi , Damarla Mahendra
Published: May 2022

Background

Apoptosis is a necessary and key pathologic feature in the development of acute lung injury (ALI). Previous research has identified MAPK Activated Protein Kinase 2 (MK2) as a potential regulator in the development of apoptosis, by facilitating the nuclear translocation of active caspase‐3, the executioner of apoptosis. Specifically, loss of MK2 results in cytoplasmic sequestration of active caspase‐3. Since MK2 is a kinase, we hypothesize that MK2 phosphorylates caspase‐3 leading to its nuclear translocation and ultimately executing apoptosis.

Methods

Caspase‐3 phosphorylation was determined by 2‐D gel electrophoresis with and without phosphatases, in addition to direct protein kinase assays using radiolabelled phosphate. Caspase‐3 localization was determined by performing nuclear and cytoplasmic extractions, and subsequent measurement of caspase‐3 activity. Apoptosis was determined by flow cytometry. NCI‐H23 (non‐small cell cancer, NSLC) cells were used as a model due to their ability to resist apoptosis, as well as their ability to sequester active‐caspase‐3 in the cytoplasm.

Results

2‐D gel electrophoresis showed that the p‐19 fragment of cleaved caspase‐3, the fragment that translocates to the nucleus, has a shift in isoelectric focus that is absent with the loss of MK2. In addition, 2‐D gel analyses of lung homogenates from WT mice exposed to LPS and treated with phosphatase have similar isoelectric foci for caspase 3 as lung homogenates from MK2‐/‐ mice exposed to LPS. Furthermore, phosphor‐imaging shows that MK2 directly phosphorylates caspase‐3. Interestingly, immunoblots of NCI‐H23 cells (which sequester active‐caspase‐3 in the cytoplasm) have lower MK2 expression than the small‐cell lung cancer cell line, H446. Flow cytometry with DAPI staining demonstrated increased apoptosis following etoposide treatment in cells over‐expressing MK2 compared to control transfections. Over‐expression of MK2 led to an increase in nuclear caspase‐3 activity. Additionally, overexpression of nuclear localization sequence (NLS) tagged caspase 3 led to increased etoposide induced apoptosis compared to WT caspase 3 or control transfections.

Conclusion

Combined these results strongly suggest that MK2 phosphorylates caspase‐3, which may facilitate the nuclear translocation of caspase‐3, thereby promoting the execution of apoptosis. Further work is needed to understand the mechanisms by which MK2 regulates the trafficking of active caspase‐3.
Doxorubicin Induces Necroptosis in Young Mice
Authors: Imaralu Omonzejie , Singla Dinender
Published: May 2022

Despite its potency as a chemotherapeutic drug, the prevalence of cardiotoxicity resulting from doxorubicin treatment in childhood survivors of cancer is a major concern. While the occurrence of apoptosis has been well ascertained in doxorubicin induced cardiotoxicity (DIC) by several studies; however, the presence of necroptosis in young DIC remains unknown. We hypothesize that doxorubicin will induce necroptotic cell death and adverse cardiac remodeling in young mice. Therefore, in this study we investigated the possible occurrence of necroptotic cell death in DIC in young mice in‐vivo to delineate underlying pathological mechanism.

C57BL6 mice (6 + 2 weeks of age) were divided into two groups: Control (mice were given 0.9% saline) and Dox (drug was administered at 2.5mg/kg body weight (BW) for six i.p injection, with a cumulative dose of 15mg/kg BW). At day 14, heart function was examined using echocardiography, mice were euthanized. Heart tissues were collected and analyzed for necroptosis, adverse cardiac remodeling using immunohistochemistry, and histology. Upon analysis, we observed that Dox treatment significantly (p<0.05) increased the expression of necroptotic markers [Receptor interacting Serine/Threonine kinase 1 and 3 (RIPK1, RIPK3) and Mixed Lineage kinase domain‐Like (MLKL)] as confirmed by immunohistochemistry. Furthermore, histological analysis showed adverse cardiac remodeling with significant increase (p<0.05) in cardiac hypertrophy and fibrosis in Dox group compared to control. Moreover, a significant decrease (p<0.05) in heart function was observed in Dox treated animals compared to control. In conclusion the results of our study suggest that doxorubicin, induces necroptotic cell death which further leads to adverse cardiac remodeling in Dox treated young mice.
Metabolic effect of orchidectomy on C57BL6/J mice exposed to intermittent hypoxia
Authors: Ganouna‐Cohen Gauthier , Blachot Minassian Britanny , Marcouiller François , Joseph Vincent
Published: May 2022

Introduction

Sleep apnea (SA) is characterized by obstructions of the airways or cessation of breathing during sleep leading to intermittent hypoxia (IH). IH is a source of oxidative stress and inflammation altering the functions of pancreas, liver, muscles and adipose tissue. As a consequence, sleep apnea patients have a high prevalence of insulin resistance and glucose intolerance which can lead to type 2 diabetes. In mice, exposures to IH induce adipose tissue inflammation, liver injury and worsen the pancreatic β‐cell injury in a model of diabetes. While these effects are associated with insulin resistance, glucose tolerance is improved, notably via an enhanced glucose uptake by skeletal muscles. In men, sleep apnea and diabetes are also characterized by low testosterone levels. Interestingly, testosterone levels are positively corelated with insulin sensitivity in human, and low testosterone levels can be associated with inflammation, oxidative stress and insulin resistance. Therefore, we tested the hypothesis that testosterone modulates the metabolic alterations induced by IH in male mice.

Method

We used intact (Sham) or orchidectomized (ORX) C57BL/6J male mice exposed for 14 days to IH (12h / day, 10 cycles / h, 6% nadir oxygen) or normoxia (Nx). The animals were weighed, and we assessed body composition in terms of lean and fat mass, as well as fluid content by using a small animal MRI scanner before the surgery, before IH or Nx exposures and at the end of the protocol. The day after the last IH session, mice were fasted for 6h, we measured fasting glycaemia and insulinemia then we performed a glucose tolerance test.

Results

In Sham mice, IH decreased bodyweight and fat mass. This was associated with a decrease of fasting glycaemia, insulinemia and HOMA‐IR (index of insulin resistance), and improved glucose sensitivity. ORX decreased bodyweight and lean tissue mass, associated with lower fasting glycemia, insulinemia, HOMA‐IR and improved whole body glucose sensitivity. ORX mice exposed to IH had a decrease of both fat and lean tissue mass leading to further reduction of body weight. However, there was no additive effects of ORX and ORX IH for fasting glycaemia, insulinemia, HOMA‐IR and glucose sensitivity.

Conclusion

IH and ORX respectively reduce fat and lean tissue mass, and these effects are additive when both stimuli are applied simultaneously. Contrastingly, there are no additive effects of IH and ORX on the metabolic parameters reported here.
Changes in oxygen and carbon dioxide in umbilical cord arterial blood before and after the first breath of a newborn to explore the mechanisms of respiratory regulation
Authors: Shi Chao , Sun Xingguo
Published: May 2022

The fetus has no actual respiration, and the newborn begins to breathe after birth. We assume that the first breath dominantly generated by hypoxia. In this study, the changes and lowest limit of blood oxygen partial pressure of umbilical artery (PuaO2) after chemoreceptor were analyzed to explore the mechanism of neonatal spontaneous breathing.

Methods

With signed consent form by all fetal parents before birth, 14 newborns successfully completed the umbilical artery or vein catheterization and drawn blood according to the heartbeat. All blood samples analyzed by blood gas analyzer, calculated and analyzed the similarities and differences between umbilical vein(Puv) and umbilical artery(Pua).

Results

Although we completed 14 newborns, there were only 9 cases of umbilical artery samples and 8 cases of umbilical vein samples were collected. Only 3 cases collected both Pua and Puv blood samples at the same tim (see serial paper II). PuaO2 in gradually decreased with time (heartbeat frequency), until Pua contracted after spontaneous breathing produced about 8‐10 heartbeats, and then could not get enough blood samples. Only 3 newborns were able to take blood samples after spontaneous breathing for 8‐10 heartbeats, and PuaO2 jumped to 93.8,137.0 and 93.8 respectively The mean value of PuaO2 was 25.94±6.79 (range 18.04‐37.55)mmHg, the highest value was 32.41±11.97 (range 23.0‐61.0) mmHg, and the lowest value was 22.18±6.35 (range 14.0‐33.6)mmHg. Although PuvO2 decreased gradually with time (heartbeat) too, most of them also showed the tendency of alternately rising and falling with the regularity of mother’s respiration. The mean value of PuvO2 was 50.35±21.35 (range 32.56‐100.73)mmHg, the highest value was 90.34±8.44 (range 43.4‐153.0)mmHg, and the lowest value was 36.96±14.90 (range 24.8‐73.8)mmHg, although.

Conclusion

PuaO2 decreases gradually with time before spontaneous breathing after the delivered fetus as a newborn, and it induces the first inhalation to start spontaneous breathing when it reaches the threshold of triggering breathing.