Variable effects of captivity on microbiomes in populations of IUCN-endangered Blanding's turtles (Emydoidea blandingii).

AIMS: Microbiome composition is increasingly considered in species reintroduction efforts and may influence survival and reproductive success. Many turtle species are threatened by anthropogenic pressures and are frequently raised in captivity for reintroduction efforts, yet little is known about turtle microbiome composition in either wild or captive settings. Here, we investigated trends in microbiome composition of captive and wild IUCN-endangered Blanding's turtles (Emydoidea blandingii). METHODS AND RESULTS: We amplified and sequenced the V4 region of the 16S rDNA locus from plastron, cloaca, and water samples of wild E. blandingii adults and two populations of captive E. blandingii juveniles being raised for headstarting. Plastron, cloaca, and water-associated microbiomes differed strongly from each other and were highly variable among captive sites and between captive and wild sites. Across plastron, cloaca, and water-associated microbial communities, microbial diversity changed over time, but not in a predictable direction between captive sites. Plastron beta diversity correlated with growth rate in captive samples, indicating that external microbiomes may correlate with individual fitness. CONCLUSIONS: Our results indicate that external and internal microbiomes vary between captive and wild turtles and may reflect differences in fitness of captive-raised individuals.

Making an Impact with E.M.P.A.C.T. Engage, Mentor Prepare, Advocate, Cultivate, and Teach: An Innovative Pilot Mentoring Program Evaluation for Students Underrepresented in Medicine.

Purpose: Medicine has yet to increase the representation of historically excluded persons in medicine to reflect the general population. The lack of support and guidance in the medical training of these individuals is a significant contributor to this disparity. The Engage, Mentor, Prepare, Advocate for, Cultivate, and Teach (EMPACT) Mentoring program was created to address this problem by providing support for learners who are historically underrepresented in medicine (URiM) as they progress through medical school. Methods: The EMPACT Pilot Program was formed and conducted during the 2019-2020 academic year. A total of 19 EMPACT mentorship groups were created, each consisting of two mentors and four medical student mentees. Additionally, four professional development workshops were held along with a final Wrap-up and Awards event. Pre and post pilot program surveys along with surveys after each workshop and focus groups were conducted with a random selection of program participants. Results: When compared to data from before and after the implementation of the EMPACT program, there were statistically significant differences (p < 0.05) in EMPACT mentees reporting they agree or strongly agree they felt ready to handle their clinical rotations (28% to 65%), felt the need to have an advocate (85% to 47%), possessed insight on day-to-day activities of an attending (26% to 56%) and felt a sense of community (79% to 94%). Mentors revealed an increase in their awareness of the concepts of microaggressions and imposter phenomenon. Finally, both groups felt an increase in their support system and sense of community at the school of medicine. Conclusion: Despite COVID-19 limitations, the EMPACT program met its goals. We effectively supported URiM medical students through mentorship, networking, and community.

Metabolic flux analysis of the non-transitory starch tradeoff for lipid production in mature tobacco leaves.

The metabolic plasticity of tobacco leaves has been demonstrated via the generation of transgenic plants that can accumulate over 30% dry weight as triacylglycerols. In investigating the changes in carbon partitioning in these high lipid-producing (HLP) leaves, foliar lipids accumulated stepwise over development. Interestingly, non-transient starch was observed to accumulate with plant age in WT but not HLP leaves, with a drop in foliar starch concurrent with an increase in lipid content. The metabolic carbon tradeoff between starch and lipid was studied using 13CO2-labeling experiments and isotopically nonstationary metabolic flux analysis, not previously applied to the mature leaves of a crop. Fatty acid synthesis was investigated through assessment of acyl-acyl carrier proteins using a recently derived quantification method that was extended to accommodate isotopic labeling. Analysis of labeling patterns and flux modeling indicated the continued production of unlabeled starch, sucrose cycling, and a significant contribution of NADP-malic enzyme to plastidic pyruvate production for the production of lipids in HLP leaves, with the latter verified by enzyme activity assays. The results suggest an inherent capacity for a developmentally regulated carbon sink in tobacco leaves and may in part explain the uniquely successful leaf lipid engineering efforts in this crop.

Quantification of Acyl-Acyl Carrier Proteins for Fatty Acid Synthesis Using LC-MS/MS.

The fatty acid biosynthetic cycle is predicated on an acyl carrier protein (ACP) scaffold where two carbon acetyl groups are added in a chain elongation process through a series of repeated enzymatic steps. The chain extension is terminated by hydrolysis with a thioesterase or direct transfer of the acyl group to a glycerophospholipid by an acyltransferase. Methods for analysis of the concentrations of acyl chains attached to ACPs are lacking but would be informative for studies in lipid metabolism. We describe a method to profile and quantify the levels of acyl-ACPs in plants, bacteria and mitochondria of animals and fungi that represent Type II fatty acid biosynthetic systems. ACPs of Type II systems have a highly conserved Asp-Ser-Leu-Asp (DSLD) amino acid sequence at the attachment site for 4'-phosphopantetheinyl arm carrying the acyl chain. Three amino acids of the conserved sequence can be cleaved away from the remainder of the protein using an aspartyl protease. Thus, partially purified protein can be enzymatically hydrolyzed to produce an acyl chain linked to a tripeptide via the 4'-phosphopantetheinyl group. After ionization and fragmentation, the corresponding fragment ion is detected by a triple quadrupole mass spectrometer using a multiple reaction monitoring method. 15N isotopically labeled acyl-ACPs generated in high amounts are used with an isotope dilution strategy to quantify the absolute levels of each acyl group attached to the acyl carrier protein scaffold.

Guidance on Implementing an Oral Chemotherapy Drug Repository Program in a Medically Integrated Pharmacy Setting.

Oncology drug repository programs allow patients to donate oral chemotherapy that can be redispensed to patients in need and could ultimately reduce drug waste. Medically integrated pharmacies can serve as a platform for drug repository programs because of the integration of healthcare providers and pharmacists at one location, facilitating an effective transition from donation to redispensing. Before implementing a program, pharmacies should consider state laws regarding who can donate medications, the type of setting (including open or closed systems), as well as how to assess the quality of the medication donated, expiration dates, storing and maintenance of a separate inventory, written policies and procedures, and a priority list for dispensing medications to patients. In this article, we provide the initial steps to assist states and oncology pharmacists interested in developing a drug repository program.

Metabolic understanding of disulfide reduction during monoclonal antibody production.

The disulfide reduction of intact monoclonal antibodies (mAbs) and subsequent formation of low molecular weight (LMW) species pose a direct risk to product stability, potency, and patient safety. Although enzymatic mechanisms of reduction are well established, an understanding of the cellular mechanisms during the bioreactor process leading to increased risk of disulfide reduction after harvest remains elusive. In this study, we examined bench, pilot, and manufacturing-scale batches of two mAbs expressed in Chinese hamster ovary (CHO) cells, where harvested cell culture fluid (HCCF) occasionally demonstrated disulfide reduction. Comparative proteomics highlighted a significant elevation in glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels in a highly reducing batch of HCCF, compared to a non-reducing batch. Analysis during production cell culture showed that increased GAPDH gene and protein expression correlated to disulfide reduction risk in HCCF in every case examined. Additionally, glucose 6-phosphate dehydrogenase (G6PD) activity and an increased (≥ 300%) lactate/pyruvate molar ratio (lac/pyr) during production cell culture correlated to disulfide reduction risk, suggesting a metabolic shift to the pentose phosphate pathway (PPP). In all, these results suggest that metabolic alterations during cell culture lead to changes in protein expression and enzyme activity that in turn increase the risk of disulfide reduction in HCCF. KEY POINTS: • Bioreactor conditions resulted in reduction susceptible harvest material. • GAPDH expression, G6PD activity, and lac/pyr ratio correlated with mAb reduction. • Demonstrated role for cell metabolic changes in post-harvest mAb reduction. Graphical abstract.

Sterile Spikelets Contribute to Yield in Sorghum and Related Grasses.

Sorghum (Sorghum bicolor) and its relatives in the grass tribe Andropogoneae bear their flowers in pairs of spikelets in which one spikelet (seed-bearing or sessile spikelet [SS]) of the pair produces a seed and the other is sterile or male (staminate). This division of function does not occur in other major cereals such as wheat (Triticum aestivum) or rice (Oryza sativa). Additionally, one bract of the SS spikelet often produces a long extension, the awn, that is in the same position as, but independently derived from, that of wheat and rice. The function of the sterile spikelet is unknown and that of the awn has not been tested in Andropogoneae. We used radioactive and stable isotopes of carbon, RNA sequencing of metabolically important enzymes, and immunolocalization of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) to show that the sterile spikelet assimilates carbon, which is translocated to the largely heterotrophic SS. The awn shows no evidence of photosynthesis. These results apply to distantly related species of Andropogoneae. Removal of sterile spikelets in sorghum significantly decreases seed weight (yield) by ∼9%. Thus, the sterile spikelet, but not the awn, affects yield in the cultivated species and fitness in the wild species.

A General Method for Quantification and Discovery of Acyl Groups Attached to Acyl Carrier Proteins in Fatty Acid Metabolism Using LC-MS/MS.

Acyl carrier proteins (ACPs) are the scaffolds for fatty acid biosynthesis in living systems, rendering them essential to a comprehensive understanding of lipid metabolism. However, accurate quantitative methods to assess individual acyl-ACPs do not exist. We developed a robust method to quantify acyl-ACPs to the picogram level. We successfully identified acyl-ACP elongation intermediates (3-hydroxyacyl-ACPs and 2,3-trans-enoyl-ACPs) and unexpected medium-chain (C10:1, C14:1) and polyunsaturated long-chain (C16:3) acyl-ACPs, indicating both the sensitivity of the method and how current descriptions of lipid metabolism and ACP function are incomplete. Such ACPs are likely important to medium-chain lipid production for fuels and highlight poorly understood lipid remodeling events in the chloroplast. The approach is broadly applicable to type II fatty acid synthase systems found in plants and bacteria as well as mitochondria from mammals and fungi because it capitalizes on a highly conserved Asp-Ser-Leu-Asp amino acid sequence in ACPs to which acyl groups attach. Our method allows for sensitive quantification using liquid chromatography-tandem mass spectrometry with de novo-generated standards and an isotopic dilution strategy and will fill a gap in our understanding, providing insights through quantitative exploration of fatty acid biosynthesis processes for optimal biofuels, renewable feedstocks, and medical studies in health and disease.

Mechanistic insights into viral clearance during the chromatography steps in antibody processes by using virus surrogates.

Viral safety is required for biological products to treat human diseases, and the burden of inactivation and or virus removal lies on the downstream purification process. Minute virus of mice (MVM) is a nonenveloped parvovirus commonly used as the worst-case model virus in validation studies because of its small size and high chemical stability. In this study, we investigated the use of MVM-mock virus particle (MVP) and bacteriophage ΦX174 as surrogates for MVM to mimic viral clearance studies, with a focus on chromatography operations. Based on structural models and comparison of log reduction value among MVM, MVP, and ΦX174, it was demonstrated that MVP can be used as a noninfectious surrogate to assess viral clearance during process development in multiple chromatography systems in a biosafety level one (BSL-1) laboratory. Protein A (ProA) chromatography was investigated to strategically assess the impact of the resin, impurities, and the monoclonal antibody product on virus removal.

A survey study with assessment of esophageal screening and genetic counseling in patients with Howel-Evans syndrome.

BACKGROUND: It is important to better understand the role that environmental risk factors play on the development of esophageal cancer in Howel-Evans families. Additionally, there is little published about appropriate esophageal cancer screening practices in families genetically confirmed to have this condition. METHODS: Surveys were distributed to 47 addresses of an American family with Howel-Evans syndrome, of which 29 responded and met inclusion criteria. Data was collected about demographics, environmental risk factors, and medical history of participants. RESULTS: We report characteristics of family members with tylosis, rates of esophageal cancer, rates of genetic counseling, and levels of environmental risk factors. Of the survey respondents, 43% reported features of tylosis, 71.4% were male and 28.6% were female and 28.6% reported leukoplakia. Only 21.4% of tylotic family members smoked, 65% drank alcohol, and 28.6% drank well water. More than half (57.1%) of the tylotic individuals had never had an esophagogastroduodenoscopy (EGD) and no one had been diagnosed with esophageal carcinoma. Only 3.4% of respondents had ever received genetic testing for Howel-Evans syndrome, despite genetic confirmation of their relatives. CONCLUSIONS: We encourage dermatologists to discuss smoking-cessation, genetic counseling, and early EGD with affected families.

Comprehensive Review of Tissue Adhesives.

BACKGROUND: Cyanoacrylates, also known as tissue adhesives or skin glues, are commonly used as sealants for lacerations and incisions and have found utility in excisional and cosmetic surgeries in both outpatient and operating room settings. OBJECTIVE: To review the surgical literature on the utilities, advantages, disadvantages, and special uses of cyanoacrylates applicable to dermatology. MATERIALS AND METHODS: PubMed was reviewed for relevant articles related to cyanoacrylates and their use in skin closures. Articles unrelated to cutaneous closures were excluded. RESULTS: Tissue adhesives polymerize to a water resistant, pliable film after application to approximated wound edges and have antibacterial properties. Adhesives slowly slough off as the wound heals, typically after 5 days. Compared with 5-0 nonabsorbable suture, adhesives provide similar tensile strength and similar patient and surgeon satisfaction. Although slightly more expensive than sutures, tissue adhesives obviate the need for wound dressings and suture removal. They do not perform as well as sutures for wounds under higher tension or in the setting of moisture and inadequate hemostasis. CONCLUSION: Cyanoacrylates serve as a safe and effective suture alternative in appropriate dermatologic surgeries and procedures.

Reliability and Validity of a Self-paced Cardiopulmonary Exercise Test in Post-MI Patients.

A self-paced peak oxygen uptake (V̇O2peak) test (SPV) has been shown to produce higher V̇O2peak values compared to standard cardiopulmonary exercise tests (sCPET), but has not been tested on any clinical population. This study aimed to assess the reliability of the SPV in a healthy population (study 1), and the validity and reliability of the SPV in post-myocardial infarction (post-MI) patients (study 2). For study 1, 25 healthy participants completed 3 SPV's. For study 2, 28 post-MI patients completed one sCPET and 2 SPV's. The SPV consisted of 5×2-min stages where participants were able to self-regulate their effort by using incremental 'clamps' in ratings of perceived exertion. The sCPET consisted of a 20 W/min ramp. Results demonstrated the SPV to have a coefficient of variation for V̇O2peak of 4.7% for the healthy population, and 8.2% for the post-MI patients. Limits of agreement ranged between±4.22-5.86 ml·kg-1·min-1, with the intraclass correlation coefficient ranging between 0.89-0.95. In study 2, there was a significantly higher V̇O2peak achieved in the SPV (23.07±4.90 ml·kg-1·min-1) against the sCPET (21.29±4.93 ml·kg-1·min-1). It is concluded that these results provide initial evidence that the SPV may be a safe, valid and reliable method for determining exercise capacity in post-MI patients.

Age differences in physiological responses to self-paced and incremental [Formula: see text] testing.

PURPOSE: A self-paced maximal exercise protocol has demonstrated higher [Formula: see text] values when compared against traditional tests. The aim was to compare physiological responses to this self-paced [Formula: see text] protocol (SPV) in comparison to a traditional ramp [Formula: see text] (RAMP) protocol in young (18-30 years) and old (50-75 years) participants. METHODS: Forty-four participants (22 young; 22 old) completed both protocols in a randomised, counter-balanced, crossover design. The SPV included 5 × 2 min stages, participants were able to self-regulate their power output (PO) by using incremental 'clamps' in ratings of perceived exertion. The RAMP consisted of either 15 or 20 W min-1. RESULTS: Expired gases, cardiac output (Q), stroke volume (SV), muscular deoxyhaemoglobin (deoxyHb) and electromyography (EMG) at the vastus lateralis were recorded throughout. Results demonstrated significantly higher [Formula: see text] in the SPV (49.68 ± 10.26 ml kg-1 min-1) vs. the RAMP (47.70 ± 9.98 ml kg-1 min-1) in the young, but not in the old group (>0.05). Q and SV were significantly higher in the SPV vs. the RAMP in the young (<0.05) but not in the old group (>0.05). No differences seen in deoxyHb and EMG for either age groups (>0.05). Peak PO was significantly higher in the SPV vs. the RAMP in both age groups (<0.05). CONCLUSION: Findings demonstrate that the SPV produces higher [Formula: see text], peak Q and SV values in the young group. However, older participants achieved similar [Formula: see text] values in both protocols, mostly likely due to age-related differences in cardiovascular responses to incremental exercise, despite them achieving a higher physiological workload in the SPV.

New cases and refinement of the critical region in the 1q41q42 microdeletion syndrome.

Microdeletions of 1q41q42 have recently been classified as a syndrome. Features include significant developmental delay and characteristic dysmorphic features as well as cleft palate, clubfeet, seizures, and short stature in some individuals, with a clinical diagnosis of Fryns syndrome in two individuals with congenital diaphragmatic hernia at the severe end of the spectrum. The gene DISP1, which is involved in sonic hedgehog signaling, has been proposed as a candidate for the midline defects in this syndrome. We undertook a genotype-phenotype analysis of seven previously unreported individuals with deletions of 1q41q42 that range from 777 kb to 6.87 Mb. Three of the individuals in our cohort do not display the major features of the syndrome and have more proximal deletions that only overlap with the previously described 1q41q42 smallest region of overlap (SRO) at DISP1. One individual with several features of the syndrome has a more distal deletion that excludes DISP1. The three remaining individuals have larger deletions that include the entire SRO and demonstrate features of the microdeletion syndrome. Confounding genotype-phenotype correlations, one of the small deletions involving DISP1 was inherited from a phenotypically normal parent. DISP1 haploinsufficiency may not be solely responsible for the major features of 1q41q42 microdeletion syndrome, and other genes in the SRO likely play a role in the phenotype. Additionally, some features present in a minority of individuals, such as Pelger-Huët anomaly, may be attributed to deletions of genes outside of the SRO.

Pediatric migraine: pharmacologic agents for prophylaxis.

OBJECTIVE: To identify and evaluate the data regarding medication use for migraine prophylaxis in the pediatric population. DATA SOURCES: Literature was obtained through searches in PubMed (Mid 1950s-March 2007), Iowa Drug Information Service/Web (1966-February 2007), International Pharmaceutical Abstracts (1970-February 2007), and the Cochrane Library. The terms migraine, prophylaxis, child, and children were used and cross referenced with all drug names. Reference citations from publications identified were also reviewed and included. STUDY SELECTION AND DATA EXTRACTION: Only trials that evaluated migraine headaches in the pediatric population were included. Trials including adolescent and adult populations are briefly listed, but not reviewed. Trials involving non-prescription medication were also included in the evaluation. Due to the limited information, all clinical trials, retrospective reviews, and abstracts evaluated were included in this review. DATA SYNTHESIS: Few controlled clinical trials regarding prophylaxis therapy are available. Currently, no medications are approved by the Food and Drug Administration for prophylaxis of migraines in children. Seventeen drugs were identified and included in the review. Of the drugs with available data, topiramate, valproic acid, flunarizine, amitriptyline, and cyproheptadine have shown efficacy in decreasing migraine frequency and duration in children. However, larger clinical trials are necessary to validate the utility of these agents. Conflicting data exist for propranolol and pizotifen, and additional data are needed for gabapentin, levetiracetam, zonisamide, naproxen, and trazodone. In clinical trials, nimodipine, clonidine, and natural supplements have shown a lack of efficacy versus placebo for prophylaxis of migraines in children. CONCLUSIONS: Topiramate, valproic acid, and amitriptyline have the most data on their use for prophylaxis of migraines in children. Numerous agents have limited data in this population and several agents lack efficacy. Prospective, well designed, controlled clinical trials that include quality-of-life and functional outcomes are needed for guiding therapy of migraine prophylaxis for children.