Rhomboids of Mycobacteria: Characterization Using an aarA Mutant of Providencia stuartii and Gene Deletion in Mycobacterium smegmatis

Background

Rhomboids are ubiquitous proteins with unknown roles in mycobacteria. However, bioinformatics suggested putative roles in DNA replication pathways and metabolite transport. Here, mycobacterial rhomboid-encoding genes were characterized; first, using the Providencia stuartii null-rhomboid mutant and then deleted from Mycobacterium smegmatis for additional insight in mycobacteria.

Methodology/Principal Findings

Using in silico analysis we identified in M. tuberculosis genome the genes encoding two putative rhomboid proteins; Rv0110 (referred to as “rhomboid protease 1”) and Rv1337 (“rhomboid protease 2”). Genes encoding orthologs of these proteins are widely represented in all mycobacterial species. When transformed into P. stuartii null-rhomboid mutant (ΔaarA), genes encoding mycobacterial orthologs of “rhomboid protease 2” fully restored AarA activity (AarA is the rhomboid protein of P. stuartii). However, most genes encoding mycobacterial “rhomboid protease 1” orthologs did not. Furthermore, upon gene deletion in M. smegmatis, the ΔMSMEG_4904 single mutant (which lost the gene encoding MSMEG_4904, orthologous to Rv1337, “rhomboid protease 2”) formed the least biofilms and was also more susceptible to ciprofloxacin and novobiocin, antimicrobials that inhibit DNA gyrase. However, the ΔMSMEG_5036 single mutant (which lost the gene encoding MSMEG_5036, orthologous to Rv0110, “rhomboid protease 1”) was not as susceptible. Surprisingly, the double rhomboid mutant ΔMSMEG_4904–ΔMSMEG_5036 (which lost genes encoding both homologs) was also not as susceptible suggesting compensatory effects following deletion of both rhomboid-encoding genes. Indeed, transforming the double mutant with a plasmid encoding MSMEG_5036 produced phenotypes of the ΔMSMEG_4904 single mutant (i.e. susceptibility to ciprofloxacin and novobiocin).

Conclusions/Significance

Mycobacterial rhomboid-encoding genes exhibit differences in complementing aarA whereby it''s only genes encoding “rhomboid protease 2” orthologs that fully restore AarA activity. Additionally, gene deletion data suggests inhibition of DNA gyrase by MSMEG_4904; however, the ameliorated effect in the double mutant suggests occurrence of compensatory mechanisms following deletion of genes encoding both rhomboids.     

Evaluation of in-house PCR for diagnosis of smear-negative pulmonary tuberculosis in Kampala, Uganda

ABSTRACT: BACKGROUND: Nucleic acid amplification tests (NAATs) have offered hope for rapid diagnosis of tuberculosis (TB). However, their efficiency with smear-negative samples has not been widely studied in low income settings. Here, we evaluated in-house PCR assay for diagnosis of smear-negative TB using Lowenstein-Jensen (LJ) culture as the baseline test. Two hundred and five pulmonary TB (PTB) suspects with smear-negative sputum samples, admitted on a short stay emergency ward at Mulago Hospital in Kampala, Uganda, were enrolled. Two smear-negative sputum samples were obtained from each PTB suspect and processed simultaneously for identification of MTBC using in-house PCR and LJ culture. RESULTS: Seventy two PTB suspects (35%, 72/205) were LJ culture positive while 128 (62.4%, 128/205) were PCR-positive. The sensitivity and specificity of in-house PCR for diagnosis of smear-negative PTB were 75% (95% CI 62.6-85.0) and 35.9% (95% CI 27.2-45.3), respectively. The positive and negative predictive values were 39% (95% CI 30.4-48.2) and 72.4% (95% CI 59.1-83.3), respectively, while the positive and negative likelihood ratios were 1.17 (95% CI 0.96-1.42) and 0.70 (95% CI 0.43-1.14), respectively.One hundred and seventeen LJ culture-negative suspects (75 PCR-positive and 42 PCR-negative) were enrolled for follow-up at 2 months. Of the PCR-positive suspects, 45 (60%, 45/75) were still alive, of whom 29 (64.4%, 29/45) returned for the follow-up visit; 15 (20%, 15/75) suspects died while another 15 (20%, 15/75) were lost to follow-up. Of the 42 PCR-negative suspects, 22 (52.4%, 22/42) were still alive, of whom 16 (72.7%, 16/22) returned for follow-up; 11 (26.2%, 11/42) died while nine (21.4%, 9/42) were lost to follow-up.Overall, more PCR-positive suspects were diagnosed with PTB during follow-up visits but the difference was not statistically significant (27.6%, 8/29 vs. 25%, 4/16, p = 0.9239). Furthermore, mortality was higher for the PCR-negative suspects but the difference was also not statistically significant (26.2% vs. 20% p = 0.7094). CONCLUSION: In-house PCR correlates poorly with LJ culture for diagnosis of smear-negative PTB. Therefore, in-house PCR may not be adopted as an alternative to LJ culture.     

Cytological analysis of ginseng carpel development

Panax ginseng Meyer, commonly known as ginseng, is considered one of the most important herbs with pharmaceutical values due to the presence of ginsenosides and is cultivated for its highly valued root for medicinal purposes. Recently, it has been recognized that ginseng fruit contains high contents of triterpene such as ginsenoside Re as pharmaceutical compounds. However, it is unclear how carpel, the female reproductive tissue of flowers, is formed during the three-year-old growth before fruit is formed in ginseng plants. Here, we report P. ginseng carpel development at the cytological level, starting from the initial stage of ovule development to seed development. The carpel of P. ginseng is composed of two free stigmas, two free styles, and one epigynous bilocular ovary containing one ovule in each locule. Based on our cytological study, we propose that the female reproductive development in P. ginseng can be classified into seven stages: early phase of ovule development, megasporogenesis, megagametogenesis, pre-fertilization, fertilization, post-fertilization, and seed development. We also describe the correlation of the female and male gametophyte development and compare morphological differences in carpel development between ginseng and other higher plants. One unique feature for ginseng seed development is that it takes 40 days for the embryo to develop to the early torpedo stage and that the embryo is small relative to the seed size, which could be a feature of taxonomic importance. This study will provide an integral tool for the study of the reproductive development and breeding of P. ginseng.     

Reference Equation for Respiratory Pressures in Pediatric Population: A Multicenter Study

Previous studies have proposed only one prediction equation for respiratory muscle strength without taking into consideration differences between ages in pediatric population. In addition, those researches were single-center studies. The objective of this study was to establish reference equations for maximal inspiratory pressure (PImax) and maximal expiratory pressure (PEmax) in children and teenagers. In a multicenter study, 450 healthy volunteers were evaluated (aged 6–18yrs). There were included volunteers with normal lung function. We excluded volunteers who could not perform the tests; participated in physical activity more than twice a week; were born prematurely; smokers; chronic respiratory, cardiologic, and/or neurologic diseases; had acute respiratory disease during the prior three weeks. The volunteers were divided into two groups: Group 6–11 (6–11yrs) and Group 12–18 (12–18yrs). PImax and PEmax were measured according to statement. The mean PImax value was 85.6 (95%IC 83.6–87.6 cmH₂O), and PEmax 84.6 (95%IC 85.5–86.2 cmH₂O). The prediction equations for PImax and PEmax for Group 6–11 were 37.458–0.559 + (age * 3.253) + (BMI * 0.843) + (age * gender * 0.985); and 38.556 + 15.892 + (age * 3.023) + (BMI * 0.579) + (age * gender * 0.881), respectively (R² = 0.34 and 0.31, P<0.001). The equations for Group 12–18 were 92.472 + (gender * 9.894) + 7.103, (R² = 0.27, P = 0.006) for PImax; and 68.113 + (gender * 17.022) + 6.46 + (BMI * 0.927), (R² = 0.34, P<0.0001) for PEmax. This multicenter study determined the respiratory muscle strength prediction equations for children and teenagers.     

Nest Maintenance Activity of <Emphasis Type="Italic">Dinoponera quadriceps</Emphasis> in a Natural Environment

In social insects, task allocation can be more complex than workers merely falling into discrete task groups. Any activity performed by the colony cannot be fully understood in isolation from other activities because they may be interrelated. Investigating activities other than foraging is crucial to understanding the global functioning and organization of ant colonies. This study attempts to characterize the nest maintenance activity of the ponerine queenless ant, Dinoponera quadriceps, in its natural environment to determine the effects of environmental variables on the variations in both seasonal and daily rhythms and to discuss its differences and possible relationships to foraging. Four colonies of D. quadriceps were observed in an area of Atlantic Forest in northeastern Brazil. Data collection was performed over a period of 72 h every three months during an entire annual cycle. Nest maintenance activity in D. quadriceps colonies was observed during both the light and dark phases of the day. There was no significant difference between the day phases in the number of workers involved in this task. On the other hand, D. quadriceps colonies exhibited seasonal variation in nest maintenance activity, peaking in the early rainy season. The seasonal rhythm of nest maintenance was positively correlated with relative humidity and negatively correlated with prey availability and rainfall. Our results indicate the existence of an annual variation in the nest maintenance activity of D. quadriceps associated with environmental variables. However, it occurs equally both at night and day, countering the hypothesis that there is a daily rhythm.     

Phenological stages of arrowroot (Maranta arundinacea L.) according to the Biologische Bundesanstalt Bundessortenamt und Chemische Industrie scale

The present study aimed to characterise and establish the phenological stages of arrowroot (Maranta arundinacea L.) by employing the extended BBCH (Biologische Bundesanstalt Bundessortenamt und Chemische Industrie) scale system and reporting a detailed biometric evaluation. An experiment with individual plants grown in separate pots was set up in a greenhouse for the identification and characterisation of phenological stages (the phenotypic characterisation and tuberisation process was accompanied by histological analyses every 2 months) and for biometric evaluation (plant height, root volume, dry biomass, leaf area and accumulated starch). We identified 36 secondary stages within eight principal stages of arrowroot growth which comprise: (0) sprouting; (1) leaf development–main stem; (2) formation of lateral stems; (3) elongation–main stem; (4) rhizome formation and tuberisation; (5) inflorescence emergence; (6) flowering, and (9) senescence. Results were categorised and recorded by way of photographs and technical drawings. The total growth cycle in this experiment was 230 days, concluding at the point of harvest. It was possible to observe the phenological events that characterised the changes at each stage (colouration, flag leaf emission, inflorescence and the rhizome tuberisation process). The BBCH system was efficient in identifying arrowroot phenological data. It constitutes a tool for agricultural calendar delineation and contributes to the standardisation of research on the species.     

Adjustments in the Time,Distance and Direction of Foraging in Dinoponera quadriceps Workers

We measured individual decisions regarding the adjustments of time, distance and direction of foraging in Dinoponera quadriceps. We observed two colonies in an area of secondary Atlantic Forest, FLONA-ICMBio, in Northeastern Brazil. The workers were individually marked. We recorded the displacement of workers, their returns to the nest with and without food, the time spent searching for food, maximum and total distance, inter-trip latency and direction of trips. The time spent searching for food, maximum distance and transport velocity did not vary with food size. The previous trip success reduced the latency between foraging trips and increased the percentage of success on the next trip. However, this previous success did not demonstrate a significant variation relative to the time spent searching on the next trip or direction of search. The workers maintained an individual directional fidelity during foraging. The adjustments of these foraging variables under individual control contributed to the efficiency at the colony level. D. quadriceps is compatible with the central place theory and risk sensitivity model of behavior.     

Mitofusin-2 mediates doxorubicin sensitivity and acute resistance in Jurkat leukemia cells

Mitochondria oscillate along a morphological continuum from fragmented individual units to hyperfused tubular networks. Their position at the junction of catabolic and anabolic metabolism couples this morphological plasticity, called mitochondrial dynamics, to larger cellular metabolic programs, which in turn implicate mitochondria in a number of disease states. In many cancers, fragmented mitochondria engage the cell with the biosynthetic capacity of aerobic glycolysis in service of proliferation and progression. Chemo-resistant cancers, however, favor remodeling dynamics that yield fused mitochondrial assemblies utilizing oxidative phosphorylation (OXPHOS) through the electron transport chain (ETC). In this study, expression of Mitofusin-2 (MFN-2), a GTPase protein mediator of mitochondrial fusion, was found to closely correlate to Jurkat leukemia cell survival post doxorubicin (DxR) assault. Moreover, this was accompanied by dramatically increased expression of OXPHOS respiratory complexes and ATP Synthase, as well as a commensurate escalation of state III respiration and respiratory control ratio (RCR). Importantly, CRISPR knockout of MFN-2 resulted in a considerable decrease of doxorubicin (DxR) median lethal dose compared to a treated wildtype control, suggesting an important role of mitochondrial fusion in chemotherapy sensitivity and acute resistance.     

Nuclear ribosomal internal transcribed spacer 1 (ITS1) variation in the Anastrepha fraterculus cryptic species complex (Diptera,Tephritidae) of the Andean region

The nuclear ribosomal internal transcribed spacer 1 (ITS1) was sequenced for Anastrepha fraterculus (Wiedemann, 1830) originating from 85 collections from the northern and central Andean countries of South America including Argentina (Tucumán), Bolivia, Perú, Ecuador, Colombia, and Venezuela. The ITS1 regions of additional specimens (17 collections) from Central America (México, Guatemala, Costa Rica, and Panamá), Brazil, Caribbean Colombia, and coastal Venezuela were sequenced and together with published sequences (Paraguay) provided context for interpretation. A total of six ITS1 sequence variants were recognized in the Andean region comprising four groups. Type I predominates in the southernmost range of Anastrepha fraterculus. Type II predominates in its northernmost range. In the central and northern Andes, the geographic distributions overlap and interdigitate with a strong elevational effect. A discussion of relationships between observed ITS1 types and morphometric types is included.