Proteogenomic analysis of polymorphisms and gene annotation divergences in prokaryotes using a clustered mass spectrometry-friendly database

Precise annotation of genes or open reading frames is still a difficult task that results in divergence even for data generated from the same genomic sequence. This has an impact in further proteomic studies, and also compromises the characterization of clinical isolates with many specific genetic variations that may not be represented in the selected database. We recently developed software called multistrain mass spectrometry prokaryotic database builder (MSMSpdbb) that can merge protein databases from several sources and be applied on any prokaryotic organism, in a proteomic-friendly approach. We generated a database for the Mycobacterium tuberculosis complex (using three strains of Mycobacterium bovis and five of M. tuberculosis), and analyzed data collected from two laboratory strains and two clinical isolates of M. tuberculosis. We identified 2561 proteins, of which 24 were present in M. tuberculosis H37Rv samples, but not annotated in the M. tuberculosis H37Rv genome. We were also able to identify 280 nonsynonymous single amino acid polymorphisms and confirm 367 translational start sites. As a proof of concept we applied the database to whole-genome DNA sequencing data of one of the clinical isolates, which allowed the validation of 116 predicted single amino acid polymorphisms and the annotation of 131 N-terminal start sites. Moreover we identified regions not present in the original M. tuberculosis H37Rv sequence, indicating strain divergence or errors in the reference sequence. In conclusion, we demonstrated the potential of using a merged database to better characterize laboratory or clinical bacterial strains.     

Natural pathology of the Baboon (Papio spp.)

Background Baboons are useful animal models for biomedical research, but the natural pathology of the baboon is not as well defined as other non‐human primates. Methods A computer search for all morphologic diagnoses from baboon necropsies at the Southwest National Primate Research Center was performed and included all the natural deaths and animals euthanized for natural causes. Results A total of 10,883 macroscopic or microscopic morphologic diagnoses in 4297 baboons were documented and are presented by total incidence, relative incidence by sex and age‐group, and mean age of occurrence. The most common diagnoses in descending order of occurrence were hemorrhage, stillborn, amyloidosis, colitis, spondylosis, and pneumonia. The systems with the most diagnoses were the digestive, urogenital, musculoskeletal, and respiratory. Conclusion This extensive evaluation of the natural pathology of the baboon should be an invaluable biomedical research resource.     

Molecular phylogeny and phylogeography of free-living Bryozoa (Cupuladriidae) from both sides of the Isthmus of Panama

Genetic data were used to identify Recent species of free-living bryozoans (Cupuladriidae) from both sides of the Isthmus of Panama, and to examine their phylogenetic relationships, species richness, and population structures. An approximately 480bp fragment of the 16S mitochondrial rRNA gene was sequenced from 182 individuals from Panama, the Gulf of Mexico, and El Salvador. Ten haplotype groups (Cupuladria 4, 5, and 6; Discoporella 1, 2, 3A, 3B, 3C, 7, and 8) were identified. Genetic distances between haplotype groups (3.2-26.5%; K2P+Gamma) were 1-2 orders of magnitude greater than within groups (0.1-1.4%). Seven of the haplotype groups represent morphologically distinct species; Discoporellas 3A-C appear to be cryptic species. Phylogenetic analyses identified two pairs of transisthmian sister clades. An average divergence rate derived from other taxa suggests that Cupuladrias 4 and 5 diverged approximately 7Ma, a Discoporella 7 clade diverged from a 3A-C clade approximately 11Ma, and the 3A-C clade radiated approximately 6-4Ma; these events all predated final closure of the isthmus? 3Ma. The Caribbean side of the isthmus, with 5 species, is only marginally richer in cupuladriids than the Pacific side, with 4, but has greater phylogenetic depth. The Caribbean retains lineages stemming from a New World Miocene radiation that are not represented in the eastern Pacific; extant eastern Pacific cupuladriids share most recent common ancestry with only two of the Caribbean lineages. Species in the eastern Pacific tend to show shallow population structures, with high levels of gene flow between geographically separate populations, whereas Caribbean species tend to show deeper populations structures, with indications of restricted gene flow between Bocas del Toro/Gulf of Mosquitos and Costa Arriba/San Blas. The population structures derive from Pleistocene histories and may be of limited value in interpreting the macroevolutionary pattern, as our results provide no evidence of speciation on either side of the isthmus following closure in the late Pliocene.     

Parasite-mediated predation between native and invasive amphipods

Parasites can structure biological communities directly through population regulation and indirectly by processes such as apparent competition. However, the role of parasites in the process of biological invasion is less well understood and mechanisms of parasite mediation of predation among hosts are unclear. Mutual predation between native and invading species is an important factor in determining the outcome of invasions in freshwater amphipod communities. Here, we show that parasites mediate mutual intraguild predation among native and invading species and may thereby facilitate the invasion process. We find that the native amphipod Gammarus duebeni celticus is host to a microsporidian parasite, Pleistophora sp. (new species), with a frequency of infection of 0-90%. However, the parasite does not infect three invading species, G. tigrinus, G. pulex and Crangonyx pseudogracilis. In field and laboratory manipulations, we show that the parasite exhibits cryptic virulence: the parasite does not affect host fitness in single-species populations, but virulence becomes apparent when the native and invading species interact. That is, infection has no direct effect on G. d. celticus survivorship, size or fecundity; however, in mixed-species experiments, parasitized natives show a reduced capacity to prey on the smaller invading species and are more likely to be preyed upon by the largest invading species. Thus, by altering dominance relationships and hierarchies of mutual predation, parasitism strongly influences, and has the potential to change, the outcome of biological invasions.     

Decomposition of Avicennia marina on an iron‐smelting slag substrate

The present study, conducted near Newcastle, Australia, used a blocked analysis of variance experimental design to compare initial nutrient concentrations and decomposition rates of Avicennia marina (grey mangrove) grown on sand and rock blast furnace slag. There were no significant differences ( ANOVA ; P > 0.05) in mean initial nutrient concentrations of total C, N and P for plants grown on the sand and slag substrates. A litterbag technique was used to estimate decomposition rates. After 360 days of incubation, repeated measures analysis did not identify significant differences between the substrates for the interaction term ‘substrate × time’ or the term ‘substrate’ for percentage weight loss or for C, N and P remaining. Avicennia marina on both substrates had nutrient characteristics and decomposition rates comparable to those found in the literature. Results suggest that initial nutrient concentration and decomposition rates are not dramatically influenced by the presence of slag.     

Dietary vitamin A intake below the recommended daily intake during pregnancy and the risk of congenital diaphragmatic hernia in the offspring

BACKGROUND

Vitamin A has been related to the etiology of congenital diaphragmatic hernia (CDH). We performed a case‐control study to investigate whether maternal dietary vitamin A intake is related to CDH in the offspring.

METHODS

Thirty‐one pregnancies diagnosed with CDH and 46 control pregnancies were included during the study. After CDH diagnosis and inclusion of controls by risk set sampling, maternal vitamin A intake was investigated with a food frequency questionnaire. Serum retinol and retinol‐binding protein were determined. Univariable and multivariable logistic regression models were used to calculate risk estimates with adjustment for potential confounders.

RESULTS

We found no significant differences in the overall nutrient and vitamin A intake between case and control mothers. After stratification in body mass index (BMI) categories, case mothers with normal weight showed a lower energy adjusted vitamin A intake (685 vs. 843 μg retinol activity equivalents [RAEs] / day; p = 0.04) and a slightly lower serum retinol (1.58 vs. 1.67 μmol/L; p = 0.08) than control mothers. Vitamin A intake <800 μg retinol activity equivalents (recommended daily intake) in normal weight mothers was associated with a significantly increased CDH risk (odds ratio [OR], 7.2; 95% confidence interval [CI], 1.5–34.4; p = 0.01). Associations were not significantly different in underweight and overweight mothers.

CONCLUSIONS

In normal‐weight mothers, dietary vitamin A intake during pregnancy below the recommended daily intake is significantly associated with an increased risk of a child with CDH. This finding supports the retinoid hypothesis in human CDH, but warrants further investigation in larger study populations. Birth Defects Research (Part A), 2013. © 2013 Wiley Periodicals, Inc.     

Tuberculosis preventive treatment in people living with HIV—Is the glass half empty or half full?

Olivia Oxlade and co-authors introduce a Collection on tuberculosis preventive therapy in people with HIV infection.

The most recent World Health Organization Global Tuberculosis (TB) Report suggests that 50% of people living with HIV (PLHIV) newly enrolled in HIV care initiated tuberculosis preventive treatment (TPT) in 2019 [1]. TPT is an essential intervention to prevent TB disease among people infected with Mycobacterium tuberculosis—some 25% of the world’s population [2]. Without TPT, it is estimated that up to 10% of individuals will progress to TB disease. Among PLHIV, the prognosis is worse. Of the approximately 1.4 million annual deaths from TB, 200,000 occur among PLHIV [1], who experience TB at rates more than 30 times [3] higher than people living without HIV.In 2018, governments at the United Nations High-Level Meeting (UNHLM) on TB committed to rapid expansion of testing for TB infection and provision of TPT [4]. The goal was the provision of TPT to at least 24 million household contacts of people with TB disease and 6 million PLHIV between 2018 and 2022. However, by the end of 2019, fewer than half a million household contacts had initiated TPT, well short of the pace needed to achieve the 5-year target [1]. On the other hand, approximately 5.3 million PLHIV have initiated TPT in the past 2 years [1], with particularly dramatic increases in countries supported by the President’s Emergency Plan for AIDS Relief (PEPFAR) [5]. Globally, among PLHIV entering HIV care programs, TPT initiation rose from 36% in 2017 to 49% in 2018 and 50% in 2019 [6,7].To provide insight into scaling up TPT for PLHIV, it is important to consider each of the many steps involved in the “cascade of care” for TPT. A previous systematic review of studies in several populations receiving TPT concluded that nearly 70% of all people who may benefit from TPT were lost to follow-up at cascade of care steps prior to treatment initiation [8]. To maximize the impact of TPT for TB prevention among PLHIV, the full TPT cascade of care must be assessed to identify problems and develop targeted solutions addressing barriers at each step. Until now, these data had not been synthesized for PLHIV.In order to address important research gaps related to TPT in PLHIV such as this one, we are now presenting a Collection in PLOS Medicine on TPT in PLHIV. In the first paper in this Collection, Bastos and colleagues performed a systematic review and meta-analysis of the TPT cascade of care in 71 cohorts with a total of 94,011 PLHIV [9]. This analysis highlights key steps in the cascade where substantial attrition occurs and identifies individual-level and programmatic barriers and facilitators at each step. In stratified analyses, they found that losses during the TPT cascade were not different in high-income compared to low- or middle-income settings, nor were losses greater in centers performing tests for TB infection (tuberculin skin test [TST] or interferon gamma release assay [IGRA]) prior to TPT initiation.The net benefits of TPT could potentially be increased through greater adoption of shorter rifamycin-based TPT regimens, for which there is increasing evidence of greater safety, improved treatment completion, and noninferior efficacy, compared to isoniazid regimens. Two reviews of rifamycin-based regimens in mostly HIV–negative adults and children concluded that they were as effective for prevention of TB as longer isoniazid-based regimens, with better treatment completion and fewer adverse events [10,11]. However, safety and tolerability of TPT regimens can differ substantially between people with and without HIV, and for rifamycin-based TPT regimens, safety outcomes were actually worse in people without HIV [12], plus there can be important drug–drug interactions between rifamycin-based regimens and antiretroviral drugs [13]. Reviews of studies focused on PLHIV concluded that TPT (regardless of regimen selected) significantly reduced TB incidence [14] and that the benefits of continuous isoniazid in high TB transmission settings outweighed the risks [15]. As part of this Collection, Yanes-Lane and colleagues conducted a systematic review and network meta-analysis of 16 randomized trials to directly and indirectly compare the risks and benefits of isoniazid and rifamycin-based TPT regimens among PLHIV [16]. Their findings highlight the better safety, improved completion, and evidence of efficacy, particularly reduced mortality, with rifamycin-based TPT regimens, while also noting improved TB prevention with extended duration mono-isoniazid regimens. Their review also revealed that few studies exist on some important at-risk populations, such was pregnant women and those with drug-resistant TB infection.In North America, recommendations changed in 2020 to favor shorter rifamycin-based regimens over isoniazid [17], but WHO still favors isoniazid [18], largely due to the lower drug costs. Although drug costs for rifamycins are typically higher than for isoniazid, their shorter duration and better safety profile mean that total costs for care (including personnel costs) may be lower for rifamycin-based regimens, even in underresourced settings [19]. The cost-effectiveness of different TPT regimens among PLHIV in underresourced settings remains uncertain, as well as the impact of antiretroviral therapy (ART), and the use of diagnostic tests for TB infection, such as TST or IGRA on cost efficiency. Uppal and colleagues, in the third paper in this Collection, performed a systematic review and meta-analysis of 61 published cost-effectiveness and transmission modeling studies of TPT among PLHIV [20]. In all studies, TPT was consistently cost-effective, if not cost saving, despite wide variation in key input parameters and settings considered.When comparing access to TPT among PLHIV to household contacts, many would consider the glass is half full, given that almost half of all PLHIV newly accessing care initiated TPT in 2018 and 2019, and the UNHLM goal of 6 million PLHIV initiating TPT was already nearly achieved by the end of 2020. This remarkable achievement is the result of strong recommendations from WHO for TPT among PLHIV for nearly a decade and strong donor support. These policies are, in turn, based on clear and consistent evidence of individual benefits from multiple randomized trials, plus consistent evidence of cost-effectiveness from many economic analyses as summarized in the papers in this Collection. These are useful lessons for scaling up TPT for other target populations, particularly household contacts, of whom less than half a million have initiated TPT, of the 24 million–person target set in 2018.However, the glass of TPT among PLHIV is also half empty. In contrast to the “90-90-90” targets, 50% of PLHIV newly enrolled in care do not initiate TPT, and PLHIV still bear a disproportionate burden of TB. Programmatic scale-up of TPT continues to encounter challenges that need to be overcome in order to translate individual-level success to population-level improvement. The study by Bastos and colleagues in this Collection has identified programmatic barriers including drug stockouts and suboptimal training for healthcare workers, but it also offers useful solutions, including integration of HIV and TPT services [9]. New evidence on the success of differentiated service delivery will also be invaluable to support programmatic scale-up in different settings [21]. Acting on this evidence will be essential to achieve the goal of full access to effective, safe, and cost-effective TPT for PLHIV.     

Trichinella spiralis: comparison of stages in host intestine with those of an Arctic Trichinella sp

The intestinal phase of Trichinella spiralis and of Trichinella sp. isolated in the Arctic were compared in experimental animals. Reproductive capacity, pathogenicity, distribution, and persistence of adults in the small intestine, morphological measurements, and release of newborn larvae in vitro were examined. Numerous passages of 40 days each for T. spiralis and the Trichinella sp. isolate in mice did not affect reproductive capacity, distribution of adults in the small intestine, and size of worms. Reproductive capacity index for T. spiralis, I = 151.27 ± 27.30 was significantly higher compared to the Trichinella sp. isolate index, I = 63.46 ± 19.34. The Trichinella sp. isolate was more pathogenic to mice and wild rodents compared to T. spiralis during the intestinal phase. Both parasites were located in the anterior part of the small intestine but the position of T. spiralis adults (P = 17.08) differed from position of the Trichinella sp. isolate adults (P = 23.46) in the small intestine. Intestinal phase of T. spiralis was longer (20 days) compared to the Trichinella sp. isolate (15 days) but sex ratios (:♂) were similar for both parasites. T. spiralis females released significantly higher numbers of larvae in vitro/24 hr compared to the Trichinella sp. isolate. Release of larvae was continuous during the intestinal phase and the average fecundity for T. spiralis was 335 larvae/female and for the Trichinella sp. isolate 114 larvae/female. Adults of T. spiralis and the Trichinella sp. isolate were morphologically indistinguishable and did not differ in size. A comparative index for the intestinal phase is proposed for comparison of any Trichinella spp. isolates and standard T. spiralis.