Tools for analysis and conditional deletion of subsets of sensory neurons [version 1; peer review: 4 approved]

Background: Somatosensation depends on primary sensory neurons of the trigeminal and dorsal root ganglia (DRG). Transcriptional profiling of mouse DRG sensory neurons has defined at least 18 distinct neuronal cell types. Using an advillin promoter, we have generated a transgenic mouse line that only expresses diphtheria toxin A (DTA) in sensory neurons in the presence of Cre recombinase. This has allowed us to ablate specific neuronal subsets within the DRG using a range of established and novel Cre lines that encompass all sets of sensory neurons. // Methods: A floxed-tdTomato-stop-DTA bacterial artificial chromosome (BAC) transgenic reporter line (AdvDTA) under the control of the mouse advillin DRG promoter was generated. The line was first validated using a Nav1.8Cre and then crossed to CGRPCreER (Calca), ThCreERT2, Tmem45bCre, Tmem233Cre, Ntng1Cre and TrkBCreER (Ntrk2) lines. Pain behavioural assays included Hargreaves’, hot plate, Randall-Selitto, cold plantar, partial sciatic nerve ligation and formalin tests. // Results: Motor activity, as assessed by the rotarod test, was normal for all lines tested. Noxious mechanosensation was significantly reduced when either Nav1.8 positive neurons or Tmem45b positive neurons were ablated whilst acute heat pain was unaffected. In contrast, noxious mechanosensation was normal following ablation of CGRP-positive neurons but acute heat pain thresholds were significantly elevated and a reduction in nocifensive responses was observed in the second phase of the formalin test. Ablation of TrkB-positive neurons led to significant deficits in mechanical hypersensitivity in the partial sciatic nerve ligation neuropathic pain model. // Conclusions: Ablation of specific DRG neuronal subsets using the AdvDTA line will be a useful resource for further functional characterization of somatosensory processing, neuro-immune interactions and chronic pain disorders

Targeted therapy against Bcl-2-related proteins in breast cancer cells

INTRODUCTION: Bcl-2 and Bcl-xL confer resistance to apoptosis, thereby reducing the effectiveness of chemotherapy. We examined the relationship between the expression of Bcl-2 and Bcl-xL and chemosensitivity of breast cancer cells, with the aim of developing specific targeted therapy. METHODS: Four human breast cancer cell lines were examined, and the effects of antisense (AS) Bcl-2 and AS Bcl-xL phosphorothioate oligodeoxynucleotides (ODNs) on chemosensitivity were tested in vitro and in vivo. Chemosensitivity was evaluated by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay, and the antitumor effect was assessed in vivo by the success of xenograft transplantation into athymic mice. RESULTS: Treatment with AS Bcl-2 and Bcl-xL ODNs resulted in a sequence-specific decrease in protein expression, compared with controls. Treatment of BT-474, ZR-75-1, and MDA-MB-231 cells with AS Bcl-2 increased chemosensitivity to doxorubicin (DOX), mitomycin C (MMC), paclitaxel (TXL), and docetaxel (TXT). Transfection of the Bcl-2 gene into MDA-MB-453 cells decreased sensitivity to DOX and MMC. Treatment of MDA-MB-231, BT-474, and ZR-75-1 cells with AS Bcl-xL increased chemosensitivity to DOX, MMC and taxanes to a smaller extent than AS Bcl-2. This occurred in the setting of increased Bax and cleaved poly(ADP-ribose) polymerase, as well as decreased Bcl-2 and pAkt. AS Bcl-2 ODNs induced splenomegaly in association with increased serum IL-12, which was attenuated by methylation of the CpG motifs of AS Bcl-2; however, methylated CpG failed to negate the increased antitumor effect of AS Bcl-2. Bcl-2 and Bcl-xL, to a smaller extent, are major determinants of chemosensitivity in breast cancer cells. CONCLUSION: Targeted therapy against Bcl-2 protein with the use of AS ODNs might enhance the effects of chemotherapy in patients with breast cancer

Recognition, Perceptions and Treatment Practices for Severe Malaria in Rural Tanzania: Implications for Accessing Rectal Artesunate as a Pre-Referral

OBJECTIVES: Preparatory to a community trial investigating how best to deliver rectal artesunate as pre-referral treatment for severe malaria; local understanding, perceptions of signs/symptoms of severe malaria and treatment-seeking patterns for and barriers to seeking biomedical treatment were investigated. METHODOLOGY/PRINCIPAL FINDINGS: 19 key informant interviews, 12 in-depth interviews and 14 focus group discussions targeting care-givers, opinion leaders, and formal and informal health care providers were conducted. Monthly fever episodes and danger signs or symptoms associated with severe malaria among under-fives were recorded. Respondents recognized convulsions, altered consciousness and coma, and were aware of their risks if not treated. But, these symptoms were perceived to be caused by supernatural forces, and traditional healers were identified as primary care providers. With some delay, mothers eventually visited a health facility when convulsions were part of the illness, despite pressures against this. Although vomiting and failure to eat/suck/drink were associated with malaria, they were not considered as indicators of danger signs unless combined with another more severe symptom. Study communities were familiar with rectal application of medicines. CONCLUSIONS/SIGNIFICANCE: Communities' recognition and awareness of major symptoms of severe malaria could encourage action, but perceptions of their causes and poor discrimination of other danger signs – vomiting and failure to feed – might impede early treatment. An effective health education targeting parents/guardians, decision-makers/advisors, and formal and informal care providers might be a prerequisite for successful introduction of rectal artemisinins as an emergency treatment. Role of traditional healers in delivering such medication to the community should be explored

Brazilian adolescents' knowledge and beliefs about abortion methods: A school-based internet inquiry

Background: Internet surveys that draw from traditionally generated samples provide the unique conditions to engage adolescents in exploration of sensitive health topics.Methods: We examined awareness of unwanted pregnancy, abortion behaviour, methods, and attitudes toward specific legal indications for abortion via a school-based internet survey among 378 adolescents aged 12-21 years in three Rio de Janeiro public schools.Results: Forty-five percent knew peers who had undergone an abortion. Most students (66.0%) did not disclose abortion method knowledge. However, girls (aOR 4.2, 95% CI 2.4-7.2), those who had experienced their sexual debut (aOR1.76, 95% CI 1.1-3.0), and those attending a prestigious magnet school (aOR 2.7 95% CI 1.4-6.3) were more likely to report methods. Most abortion methods (79.3%) reported were ineffective, obsolete, and/or unsafe. Herbs (e.g. marijuana tea), over-the-counter medications, surgical procedures, foreign objects and blunt trauma were reported. Most techniques (85.2%) were perceived to be dangerous, including methods recommended by the World Health Organization. A majority (61.4%) supported Brazil's existing law permitting abortion in the case of rape. There was no association between gender, age, sexual debut, parental education or socioeconomic status and attitudes toward legal abortion. However, students at the magnet school supported twice as many legal indications (2.7, SE.27) suggesting a likely role of peers and/or educators in shaping abortion views.Conclusions: Abortion knowledge and attitudes are not driven simply by age, religion or class, but rather a complex interplay that includes both social spaces and gender. Prevention of abortion morbidity and mortality among adolescents requires comprehensive sexuality and reproductive health education that includes factual distinctions between safe and unsafe abortion methods

Fecal microbiota and bile acid interactions with systemic and adipose tissue metabolism in diet-induced weight loss of obese postmenopausal women

Microbiota and bile acids in the gastrointestinal tract profoundly alter systemic metabolic processes. In obese subjects, gradual weight loss ameliorates adipose tissue inflammation and related systemic changes. We assessed how rapid weight loss due to a very low calorie diet (VLCD) affects the fecal microbiome and fecal bile acid composition, and their interactions with the plasma metabolome and subcutaneous adipose tissue inflammation in obesity. We performed a prospective cohort study of VLCD-induced weight loss of 10% in ten grades 2-3 obese postmenopausal women in a metabolic unit. Baseline and post weight loss evaluation included fasting plasma analyzed by mass spectrometry, adipose tissue transcription by RNA sequencing, stool 16S rRNA sequencing for fecal microbiota, fecal bile acids by mass spectrometry, and urinary metabolic phenotyping by H-NMR spectroscopy. Outcome measures included mixed model correlations between changes in fecal microbiota and bile acid composition with changes in plasma metabolite and adipose tissue gene expression pathways. Alterations in the urinary metabolic phenotype following VLCD-induced weight loss were consistent with starvation ketosis, protein sparing, and disruptions to the functional status of the gut microbiota. We show that the core microbiome was preserved during VLCD-induced weight loss, but with changes in several groups of bacterial taxa with functional implications. UniFrac analysis showed overall parallel shifts in community structure, corresponding to reduced abundance of the genus Roseburia and increased Christensenellaceae;g__ (unknown genus). Imputed microbial functions showed changes in fat and carbohydrate metabolism. A significant fall in fecal total bile acid concentration and reduced deconjugation and 7-α-dihydroxylation were accompanied by significant changes in several bacterial taxa. Individual bile acids in feces correlated with amino acid, purine, and lipid metabolic pathways in plasma. Furthermore, several fecal bile acids and bacterial species correlated with altered gene expression pathways in adipose tissue. VLCD dietary intervention in obese women changed the composition of several fecal microbial populations while preserving the core fecal microbiome. Changes in individual microbial taxa and their functions correlated with variations in the plasma metabolome, fecal bile acid composition, and adipose tissue transcriptome

Beneficial effects of reading aloud and solving simple arithmetic calculations (learning therapy) on a wide range of cognitive functions in the healthy elderly: study protocol for a randomized controlled trial

<p>Abstract</p> <p>Background</p> <p>Almost all cognitive functions decline with age. Results of previous studies have shown that cognitive training related to everyday life (reading aloud and solving simple arithmetic calculations), namely learning therapy, can improve two cognitive function (executive functions and processing speed) in elderly people. However, it remains unclear whether learning therapy engenders improvement of various cognitive functions or not. We investigate the impact of learning therapy on various cognitive functions (executive functions, episodic memory, short-term memory, working memory, attention, reading ability, and processing speed) in healthy older adults.</p> <p>Methods</p> <p>We use a single-blinded intervention with two parallel groups (a learning therapy group and a waiting list control group). Testers are blind to the study hypothesis and the group membership of participants. Through an advertisement in local newspaper, 64 healthy older adults are recruited. They will be assigned randomly to a learning therapy group or a waiting list control group. In the learning therapy group, participants are required to perform two cognitive tasks for 6 months: reading Japanese aloud and solving simple calculations. The waiting list group does not participate in the intervention. The primary outcome measure is the Stroop test score: a measure of executive function. Secondary outcome measures are assessments including the following: verbal fluency task, logical memory, first and second names, digit span forward, digit span backward, Japanese reading test, digit cancellation task, digit symbol coding, and symbol search. We assess these outcome measures before and after the intervention.</p> <p>Discussion</p> <p>This report is the first study which investigates the beneficial effects of learning therapy on a wide range of cognitive functions of elderly people. Our study provides sufficient evidence of learning therapy effectiveness. Most cognitive functions, which are correlated strongly with daily life activities, decrease with age. These study results can elucidate effects of cognitive training on elderly people.</p> <p>Trial registration</p> <p>This trial was registered in The University Hospital Medical Information Network Clinical Trials Registry (No. <a href="http://www.clinicaltrials.gov/ct2/show/UMIN000006998">UMIN000006998</a>).</p

COVID-19-related absence among surgeons: development of an international surgical workforce prediction model

Background: During the initial COVID-19 outbreak up to 28.4 million elective operations were cancelled worldwide, in part owing to concerns that it would be unsustainable to maintain elective surgery capacity because of COVID-19-related surgeon absence. Although many hospitals are now recovering, surgical teams need strategies to prepare for future outbreaks. This study aimed to develop a framework to predict elective surgery capacity during future COVID-19 outbreaks. Methods: An international cross-sectional study determined real-world COVID-19-related absence rates among surgeons. COVID-19-related absences included sickness, self-isolation, shielding, and caring for family. To estimate elective surgical capacity during future outbreaks, an expert elicitation study was undertaken with senior surgeons to determine the minimum surgical staff required to provide surgical services while maintaining a range of elective surgery volumes (0, 25, 50 or 75 per cent). Results Based on data from 364 hospitals across 65 countries, the COVID-19-related absence rate during the initial 6 weeks of the outbreak ranged from 20.5 to 24.7 per cent (mean average fortnightly). In weeks 7–12, this decreased to 9.2–13.8 per cent. At all times during the COVID-19 outbreak there was predicted to be sufficient surgical staff available to maintain at least 75 per cent of regular elective surgical volume. Overall, there was predicted capacity for surgeon redeployment to support the wider hospital response to COVID-19. Conclusion: This framework will inform elective surgical service planning during future COVID-19 outbreaks. In most settings, surgeon absence is unlikely to be the factor limiting elective surgery capacity

Detection and Characterization of Wolbachia Infections in Natural Populations of Aphids: Is the Hidden Diversity Fully Unraveled?

Aphids are a serious threat to agriculture, despite being a rather small group of insects. The about 4,000 species worldwide engage in highly interesting and complex relationships with their microbial fauna. One of the key symbionts in arthropods is Wolbachia, an α-Proteobacterium implicated in many important biological processes and believed to be a potential tool for biological control. Aphids were thought not to harbour Wolbachia; however, current data suggest that its presence in aphids has been missed, probably due to the low titre of the infection and/or to the high divergence of the Wolbachia strains of aphids. The goal of the present study is to map the Wolbachia infection status of natural aphids populations, along with the characterization of the detected Wolbachia strains. Out of 425 samples from Spain, Portugal, Greece, Israel and Iran, 37 were found to be infected. Our results, based mainly on 16S rRNA gene sequencing, indicate the presence of two new Wolbachia supergroups prevailing in aphids, along with some strains belonging either to supergroup B or to supergroup A

Implications of the polymorphism of HLA-G on its function, regulation, evolution and disease association

The HLA-G gene displays several peculiarities that are distinct from those of classical HLA class I genes. The unique structure of the HLA-G molecule permits a restricted peptide presentation and allows the modulation of the cells of the immune system. Although polymorphic sites may potentially influence all biological functions of HLA-G, those present at the promoter and 3′ untranslated regions have been particularly studied in experimental and pathological conditions. The relatively low polymorphism observed in the MHC-G coding region both in humans and apes may represent a strong selective pressure for invariance, whereas, in regulatory regions several lines of evidence support the role of balancing selection. Since HLA-G has immunomodulatory properties, the understanding of gene regulation and the role of polymorphic sites on gene function may permit an individualized approach for the future use of HLA-G for therapeutic purposes