Electron microscopy of human skin fibroblasts in situ during growth in culture

We have studied the electron microscopic (EM) appearance of cultured diploid human skin fibroblasts during logarithmic and confluent stages of growth using a simple technique which permits in situ visualization of individual cells in monolayer. By comparison, cells disrupted from a monolayer and pelleted showed drastic distortion of the cell surface and appearance of organelles: mechanical scraping produced massive dilatation of the rough endoplasmic reticulum (RER); and trypsin-produced multiple blebs of the plasma membrane and cytoplasmic vacuoles. In situ, these changes in trypsinized cells disappeared within 1 h after plating. Six hours later, microtubules and microfilaments had surrounded the nucleus and were oriented in longitudinal bundles beneath the plasma membrane during rapid growth. At confluence these cytoskeletal elements seemed to extend beyond the plasma membrane at intercellular junctions. Pinocytotic vesicles were abundant at those surfaces devoid of filaments. Mitochondria, aligned with the long axis of the cell, were extremely long and narrow. During logarithmic growth there were many free ribosomes, polysomes, and some flat cisternae of RER. At confluence most ribosomes were found in spirals on dilated saccules of RER which contained electron-dense material. Lysosomes of several types were present during all phases of growth and varied in number from cell to cell. Late in culture the lysosomes tended to be larger, often occupying whole areas of cytoplasm or even extruding from the cell, and resembled those seen in lysosomal storage diseases. Understanding the in situ ultrastructure of normal human fibroblasts during growth in culture will permit systematic examination of such cells in a variety of pathological states.     

Higher survival rate of vitrified and thawed in vitro produced bovine blastocysts following culture in defined medium supplemented with beta-mercaptoethanol

The present study was conducted to compare bovine embryo developmental quality, after culture in different defined culture media, up to blastocyst stage, and subsequently cultured in media supplemented with beta-mercaptoethanol (beta-ME) following blastocyst vitrification and thawing. In part one of this study, presumptive zygotes were randomly allocated into the following media: (1) CR1, (2) KSOM, (3) SOF, and (4) sequential KSOM-SOF. In the second part of the study, blastocysts derived from four different culture media were subjected to a solid surface vitrification (35% (v/v) ethylene glycol+0.5M Sucrose+5% (w/v) Polyvinylpyrrolidone (PVP), and tested for the effect of beta-ME on their post-vitrification survival. Following thawing, blastocysts were cultured with or without beta-ME. Culture medium had no effect on cleavage rates; however, a significantly greater number of zygotes cultured in KSOM, KSOM-SOF, or SOF developed to the 8-cell stage, compared with those cultured in CR1. A greater proportion of the zygotes cultured in SOF or KSOM-SOF reached blastocysts, than did those cultured in CR1 or KSOM. The use of sequential KSOM-SOF significantly increased total cell numbers of Day 7 expanded-blastocysts when compared to those cultured in CR1, KSOM, or SOF. Addition of beta-ME into culture media after vitrification and thawing improved blastocyst survival, hatching rates, and total cell numbers of blastocysts. In conclusion, supplementation of beta-ME into culture medium after vitrification and thawing significantly increased blastocyst survival, hatching rates, and their total cell numbers. These results suggest that vitrified IVF embryos should be thawed and briefly cultured in beta-ME medium prior to embryo transfer.     

Geodermatophilus chilensis sp. nov., from soil of the Yungay core-region of the Atacama Desert,Chile

A polyphasic study was undertaken to establish the taxonomic status of three representative Geodermatophilus strains isolated from an extreme hyper-arid Atacama Desert soil. The strains, isolates B12^T, B20 and B25, were found to have chemotaxonomic and morphological properties characteristic of the genus Geodermatophilus. The isolates shared a broad range of chemotaxonomic, cultural and physiological features, formed a well-supported branch in the Geodermatophilus 16S rRNA gene tree in which they were most closely associated with the type strain of Geodermatophilus obscurus. They were distinguished from the latter by BOX-PCR fingerprint patterns and by chemotaxonomic and other phenotypic properties. Average nucleotide identity, average amino acid identity and digital DNA–DNA hybridization values between the whole genome sequences of isolate B12^T and G. obscurus DSM 43160^T were 89.28%, 87.27% and 37.4%, respectively, metrics consistent with its classification as a separate species. On the basis of these data, it is proposed that the isolates be assigned to the genus Geodermatophilus as Geodermatophilus chilensis sp. nov. with isolate B12^T (CECT 9483^T = NCIMB 15089^T) as the type strain. Analysis of the whole genome sequence of G. chilensis B12^T with 5341 open reading frames and a genome size of 5.5 Mb highlighted genes and gene clusters that encode for properties relevant to its adaptation to extreme environmental conditions prevalent in extreme hyper-arid Atacama Desert soils.     

Invited review: biophysical properties of sodium channels in lung alveolar epithelial cells.

Amiloride-sensitive sodium channels in the lung play an important role in lung fluid balance. Particularly in the alveoli, sodium transport is closely regulated to maintain an appropriate fluid layer on the surface of the alveoli. Alveolar type II cells appear to play an important role in this sodium transport, with the role of alveolar type I cells being less clear. In alveolar type II cells, there are a variety of different amiloride-sensitive, sodium-permeable channels. This significant diversity appears to play a role in both normal lung physiology and in pathological states. In many epithelial tissues, amiloride-sensitive epithelial sodium channels (ENaC) are formed from three subunit proteins, designated alpha-, beta-, and gamma-ENaC. At least part of the diversity of sodium-permeable channels in lung arises from the assembling of different combinations of these subunits to form channels with different biophysical properties and different mechanisms for regulation. This leads to epithelial tissue in the lung, which has enormous flexibility to alter the magnitude and regulation of salt and water transport. In this review, we discuss the biophysical properties and occurrence of these various channels and some of the mechanisms for their regulation.     

Epidemiological,clinical, and public health response characteristics of a large outbreak of diphtheria among the Rohingya population in Cox’s Bazar,Bangladesh, 2017 to 2019: A retrospective study

BackgroundUnrest in Myanmar in August 2017 resulted in the movement of over 700,000 Rohingya refugees to overcrowded camps in Cox’s Bazar, Bangladesh. A large outbreak of diphtheria subsequently began in this population.Methods and findingsData were collected during mass vaccination campaigns (MVCs), contact tracing activities, and from 9 Diphtheria Treatment Centers (DTCs) operated by national and international organizations. These data were used to describe the epidemiological and clinical features and the control measures to prevent transmission, during the first 2 years of the outbreak. Between November 10, 2017 and November 9, 2019, 7,064 cases were reported: 285 (4.0%) laboratory-confirmed, 3,610 (51.1%) probable, and 3,169 (44.9%) suspected cases. The crude attack rate was 51.5 cases per 10,000 person-years, and epidemic doubling time was 4.4 days (95% confidence interval [CI] 4.2–4.7) during the exponential growth phase. The median age was 10 years (range 0–85), and 3,126 (44.3%) were male. The typical symptoms were sore throat (93.5%), fever (86.0%), pseudomembrane (34.7%), and gross cervical lymphadenopathy (GCL; 30.6%). Diphtheria antitoxin (DAT) was administered to 1,062 (89.0%) out of 1,193 eligible patients, with adverse reactions following among 229 (21.6%). There were 45 deaths (case fatality ratio [CFR] 0.6%). Household contacts for 5,702 (80.7%) of 7,064 cases were successfully traced. A total of 41,452 contacts were identified, of whom 40,364 (97.4%) consented to begin chemoprophylaxis; adherence was 55.0% (N = 22,218) at 3-day follow-up. Unvaccinated household contacts were vaccinated with 3 doses (with 4-week interval), while a booster dose was administered if the primary vaccination schedule had been completed. The proportion of contacts vaccinated was 64.7% overall. Three MVC rounds were conducted, with administrative coverage varying between 88.5% and 110.4%. Pentavalent vaccine was administered to those aged 6 weeks to 6 years, while tetanus and diphtheria (Td) vaccine was administered to those aged 7 years and older. Lack of adequate diagnostic capacity to confirm cases was the main limitation, with a majority of cases unconfirmed and the proportion of true diphtheria cases unknown.ConclusionsTo our knowledge, this is the largest reported diphtheria outbreak in refugee settings. We observed that high population density, poor living conditions, and fast growth rate were associated with explosive expansion of the outbreak during the initial exponential growth phase. Three rounds of mass vaccinations targeting those aged 6 weeks to 14 years were associated with only modestly reduced transmission, and additional public health measures were necessary to end the outbreak. This outbreak has a long-lasting tail, with Rt oscillating at around 1 for an extended period. An adequate global DAT stockpile needs to be maintained. All populations must have access to health services and routine vaccination, and this access must be maintained during humanitarian crises.

Jonathan Polonsky and colleagues report on a diphtheria outbreak among Rohingya people in Cox’s Bazar, Bangladesh during 2017-19.     

Biogeography and diversification of the Pacific ant genus Lordomyrma Emery

Aim This study addresses the origins of terrestrial biodiversity of the Fijian islands using the ant genus Lordomyrma (Hymenoptera: Formicidae: Myrmicinae) as a model system. We derive the evolution of the genus and determine its closest extra-Fijian relatives from geological data, molecular phylogenetic reconstruction and divergence estimates. Location Ant taxa were sampled in the Southwest Pacific, Melanesia, Southeast Asia, Australia and mainland China. Methods Phylogeny and divergence estimates of the ant genus Lordomyrma based on four nuclear genes (28S, ArgK, LW Rh, CAD) plus data on Indo-Pacific geological history are used to address current hypotheses regarding the origins of the Fijian biota. Results The genus Lordomyrma probably originated in mainland Asia, with subsequent colonization of Australia and the Pacific. The Fijian Lordomyrma clade is monophyletic, and originated c. 8.8 Ma, when it diverged from a sister group in Papua New Guinea. Main conclusions The colonization of Fiji by Lordomyrma is probably a result of long-distance dispersal from New Guinea, possibly aided by island hopping across the Vitiaz Arc. The timeline of diversification in Lordomyrma is broadly congruent with the Miocene fragmentation of the Vitiaz Arc and the Pliocene emergence of Vanua Levu. The biotic shuttle hypothesis, which posits ‘Eua Island as the source of Fijian endemics, is rejected based on the sister relationship of Fiji and New Guinea lineages, as well as on the Miocene submergence of the terrane below sea level. The diversity of Fijian Lordomyrma results from the radiation of a single lineage, which diverged from a New Guinea sister group. The genus appears to have originated in Asia rather than in Australia.