Evaluating Serum Calcium and Magnesium Levels as Predictive Biomarkers for Tuberculosis and COVID-19 Severity: A Romanian Prospective Study.

In Romania, the highest incidence of tuberculosis (TB) within the European Union was reported in 2020, highlighting a significant health challenge. This is compounded by the COVID-19 pandemic, which has severely impacted healthcare services, including TB management. Both TB and COVID-19, diseases with considerable morbidity and mortality, have shown potential links to electrolyte imbalances. We conducted a prospective study at Victor Babes Hospital, Romania on 146 patients (74 with TB, 72 with COVID-19) between December 2021 and July 2023. This study assessed correlations between disease severity and serum calcium and magnesium levels, as well as pulmonary function. Adult patients with confirmed diagnoses and comprehensive medical records were included, excluding those with chronic respiratory diseases or unrelated electrolyte imbalances. Statistical analysis utilized the Kruskal-Wallis test and Dunn's procedure for non-normally distributed data. Low serum calcium and magnesium levels were significantly correlated with severe forms of TB and COVID-19, suggesting their potential as biomarkers of disease progression. Patients with more severe TB (i.e., multiple cavities) exhibited significantly lower serum calcium (p = 0.0049) and magnesium levels (p = 0.0004). ROC analysis revealed high AUC values for serum calcium and serum magnesium in predicting COVID-19 severity, indicating their potential as biomarkers. This study demonstrates a significant association between lower serum calcium and magnesium levels and increased TB severity. Similarly, these electrolytes show promise as predictive markers for COVID-19 severity. These findings could serve as biomarkers for predicting the severity of TB and COVID-19, offering potential utility in clinical decision-making.

Disassembly of the apical junctional complex during the transmigration of Leptospira interrogans across polarized renal proximal tubule epithelial cells.

Bacterial pathogens have evolved multiple strategies to disassemble epithelial cell apical junctional complexes (AJCs) and infect epithelial cells. Leptospirosis is a widespread zoonotic infection, mainly caused by Leptospira interrogans, and its dissemination across host cell barriers is essential for its pathogenesis. However, the mechanism of bacterial dissemination across epithelial cell barriers remains poorly characterised. In this study, we analysed the interaction of L. interrogans with renal proximal tubule epithelial cells (RPTECs) and found that at 24 hr post-infection, L. interrogans remain in close contact with the plasma membrane of the RPTEC by extracellularly adhering or crawling. Leptospira interrogans cleaved E-cadherin and induced its endocytosis with release of the soluble N-terminal fragment into the extracellular medium. Concomitantly, a gradual decrease in transepithelial electrical resistance (TEER), mislocalisation of AJC proteins (occludin, claudin-10, ZO-1, and cingulin) and cytoskeletal rearrangement were observed. Inhibition of clathrin-mediated E-cadherin endocytosis prevented the decrease in TEER. We showed that disassembly of AJCs in epithelial cells and transmigration of bacteria through the paracellular route are important for the dissemination of L. interrogans in the host.

Characterizing interactions of Leptospira interrogans with proximal renal tubule epithelial cells.

BACKGROUND: Leptospira interrogans is a pathogenic, spirochetal bacterium that is responsible for leptospirosis, an emerging worldwide zoonosis. Leptospires colonize the renal proximal tubules and chronically infect the kidney. Live bacteria are excreted into urine, contaminating the environment. While it is well known that leptospires can persist in the kidneys without signs of disease for several months, the interactions of leptospires with the proximal renal epithelial tubule cells that allow the chronic renal colonization have not been elucidated yet. In the present study, we compared the interactions between a virulent, low passage (LP) strain and a cultured-attenuated, high passage (HP) strain with renal proximal tubule epithelial cells (RPTECs) to elucidate the strategies used by Leptospira to colonize the kidney. RESULTS: Kinetics analysis of kidney colonization in a mouse model of chronic infection performed by quantitative real-time PCR and immunofluorescence, showed that the LP strain reached the kidney by 3 days post infection (pi) and attached to the basal membrane side of the renal epithelial cells. At 10 days pi, some leptospires were attached to the luminal side of the tubular epithelia and the number of colonizing leptospires gradually increased. On the other hand, the HP strain was cleared during hematogenous dissemination and did not colonize the kidney. Transmission electron microscopy analysis of LP-infected kidneys at 25 days pi showed aggregated leptospires and membrane vesicles attached to the epithelial brush border. Leptospiral kidney colonization altered the organization of the RPTEC brush border. An in vitro model of infection using TCMK-1 cells, showed that leptospiral infection induced a host stress response, which is delayed in LP-infected cells. CONCLUSIONS: After hematogenous dissemination, leptospires create protective and replicative niches in the base membrane and luminal sides of the RPTECs. During the long-term colonization, leptospires attached to the RPTEC brush borders and membrane vesicles might be involved in the formation of a biofilm-like structure in vivo. Our results also suggested that the virulent strain is able to manipulate host cell stress responses to promote renal colonization.

Effects of Psidium guajava leaf extract on secretion systems of Gram-negative enteropathogenic bacteria.

We screened a total of 672 plant-tissue extracts to search for phytochemicals that inhibit the function of the type III secretion system (T3SS) of enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC). Among candidates examined, we found that an extract from the leaves of Psidium guajava (guava) inhibited the secretion of the EspB protein from EPEC and EHEC without affecting bacterial growth. The guava extract (GE) also inhibited EPEC and EHEC from adhering to and injecting EspB protein into HEp-2 cells. GE seemed to block the translocation of EspB from the bacterial cells to the culture medium. In addition to EPEC and EHEC, GE also inhibited the T3SS of Yersinia pseudotuberculosis and Salmonella enterica serovar Typhimurium. After exposure to GE, Y. pseudotuberculosis stopped the secretion of Yop proteins and lost its ability to induce the apoptosis of mouse bone marrow-derived macrophages. S. Typhimurium exposed to GE ceased the secretion of Sip proteins and lost its ability to invade HEp-2 cells. GE inhibited EspC secretion, the type V secretion protein of EPEC, but not Shiga toxin2 from EHEC. Thus, our results suggest that guava leaves contain a novel type of antimicrobial compound that could be used for the therapeutic treatment and prevention of gram-negative enteropathogenic bacterial infections.

The anemonin content of four different Ranunculus species.

The Ranunculus species are poorly known as medicinal plants. They have potential toxicity given by the ranunculin and its enzymatic degradation compounds: protoanemonin and anemonin. This paper aims to evaluate the anemonin content of four species: R. bulbosus, R. ficaria, R. sardous and R. sceleratus. The evaluation was performed by TLC and HPLC. There were evaluated two types of extracts hydroalcoholic (HA) and glycerol-ethanol (GE). The most concentrated extract in anemonin was found to be the R. sardous aerial part HA extract: 2.66 mg/ml. The lowest anemonin content is in R. sceleratus: 0.13-0.19 mg/ml. In R. bulbosus aerial part the anemonin content is less than the used HPLC method detection limits (7.68 mg/ml). In all cases the GE extracts are less concentrated in anemonin, being more safely for human administration.

Terpenic profile of different Rosmarinus officinalis extracts.

The Rosemary (Rosmarinus officinalis L.), a well-known medicinal and culinary herb, was studied to compare the terpenic profile of different extracts obtained from dry and fresh herb. There were studied the volatile oil extracted by hydro distillation from dry plant, the hydroalcoholic extracts obtained from fresh respectively dry plant and the glycerol macerate obtained from fresh plant, by GC-MS using headspace injection. The separated compounds were identified using a MS spectra library. The quantitative determination was performed by normalization respectively by calibration curve method for 1,8-cineole, alpha-pinene and D-limonene. The main separated compounds were alpha-pinene, 1,8-cineol, camphene, camphor, D-limonene and cymene. A significant difference was observed between the 4 samples volatile profiles. 1,8-cineole was found major component of the essential oil (VO-21.39%) and glycerol macerate (GM-35.60%), while and α-pinene was detected as the main constituent of the two tinctures (T-46.05%; MT-31.93%). The highest 1,8-cineol content, determined by calibration curve method, was found in the volatile oil, while the fresh plant hydroalcoholic extract was richer in α-pinene and D-limonene.

Vibrio parahaemolyticus effector proteins suppress inflammasome activation by interfering with host autophagy signaling.

Bacterial pathogens utilize pore-forming toxins or sophisticated secretion systems to establish infection in hosts. Recognition of these toxins or secretion system by nucleotide-binding oligomerization domain leucine-rich repeat proteins (NLRs) triggers the assembly of inflammasomes, the multiprotein complexes necessary for caspase-1 activation and the maturation of inflammatory cytokines such as IL-1ß or IL-18. Here we demonstrate that both the NLRP3 and NLRC4 inflammasomes are activated by thermostable direct hemolysins (TDHs) and type III secretion system 1 (T3SS1) in response to V. parahaemolyticus infection. Furthermore, we identify T3SS1 secreted effector proteins, VopQ and VopS, which induce autophagy and the inactivation of Cdc42, respectively, to prevent mainly NLRC4 inflammasome activation. VopQ and VopS interfere with the assembly of specks in infected macrophages. These data suggest that bacterial effectors interfere with inflammasome activation and contribute to bacterial evasion from the host inflammatory responses.

Leptospiral outer membrane protein LMB216 is involved in enhancement of phagocytic uptake by macrophages.

Leptospira interrogans is responsible for the zoonotic disease leptospirosis. The pathogenic mechanisms of this spirochaete remain poorly understood; however, virulence has been correlated with increased phagocytic uptake and survival within macrophages. Leptospiral outer membrane proteins are thought to be responsible for persistence in vivo via interaction with specific host components. In this study, we analysed the transcriptional profile of a virulent strain and its culture-attenuated derivative strain to identify bacterial factors that may be involved in pathogenesis. Two outer membrane proteins, LMB216 and LigB (leptospiral immunoglobulin-like protein B) were downregulated more than 10-fold in the culture-attenuated strain. We show that both proteins play a role in leptospiral uptake by macrophages and that LMB216, as well as LigB, enhances the binding of leptospires to fibronectin. Taken together, our results indicate that LMB216 plays a role in pathogen interaction with host molecule/s, which may contribute to pathogenesis of leptospirosis.

The Effect of Electroporation of a Lyotroic Liquid Crystal Genistein-Based Formulation in the Recovery of Murine Melanoma Lesions.

A lamellar lyotropic liquid crystal genistein-based formulation (LLC-Gen) was prepared in order to increase the aqueous solubility of the lipophilic phytocompound genistein. The formulation was applied locally, in a murine model of melanoma, with or without electroporation. The results demonstrated that, when the formulation was applied by electroporation, the tumors appeared later. During the 21 days of the experiment, the LLC-Gen formulation decreased the tumor volume, the amount of melanin and the degree of erythema, but when electroporation was applied, all these parameters indicated a better prognosis even (lower tumor volume, amount of melanin and degree of erythema). Although hematoxylin-eosin (HE) staining confirmed the above events, application of the LLC-Gen formulation by electroporation did not lead to a significant effect in terms of the serum concentrations of the protein S100B and serum neuron specific enolase (NSE), or the tissue expression of the platelet-derived growth factor receptor ß (PDGFRß) antibody.

Comparative Studies on Polyphenolic Composition, Antioxidant and Diuretic Effects of Nigella sativa L. (Black Cumin) and Nigella damascena L. (Lady-in-a-Mist) Seeds.

This study was performed to evaluate the phenolic profile, antioxidant and diuretic effects of black cumin and lady-in-a-mist seeds. In the phenolic profile, differences between the two species are significant. Qualitative and quantitative analyses of the phenolic compounds were performed using a HPLC-UV/MS method. Hyperoside was the only identified flavonoid glycoside (1.08 ± 0.01 µg∙g-1 dw plant material), in the N. damascena extract. Regarding the flavonol profile, kaempferol was identified before the hydrolysis, only in the N. sativa extract (6.06 ± 0.02 µg∙g-1 dw plant material) and quercetin only in N. damascena seeds (14.35 ± 0.02 µg∙g-1 dw plant material). The antioxidant potential of the two species was tested through several electron transfer assays, which indicated, excepting for the FRAP assay, N. damascena as exhibiting a higher free radical scavenging activity. The diuretic activity of the two extracts was tested using a rat-experimental model on acute diuresis. Administration of the ethanolic extract of N. sativa (100 mg∙kg-1) resulted in a significant increase in urine volume, although less than found with the reference drug; in addition N. damascena extract did not present a diuretic effect. In reference to the elimination of Na+, K+ and uric acid, the black cumin extract exhibited a higher natriuretic than kaluretic effect and a similar uricosuric effect with control and N. damascena. For N. damascena, the Na+/K+ ratio was sub unitary, but not due to an increasing of the kaluretic effect, but mostly to a decrease of Na+ excretion.

High-temperature protein G is an essential virulence factor of Leptospira interrogans.

Leptospira interrogans is a global zoonotic pathogen and is the causative agent of leptospirosis, an endemic disease of humans and animals worldwide. There is limited understanding of leptospiral pathogenesis; therefore, further elucidation of the mechanisms involved would aid in vaccine development and the prevention of infection. HtpG (high-temperature protein G) is the bacterial homolog to the highly conserved molecular chaperone Hsp90 and is important in the stress responses of many bacteria. The specific role of HtpG, especially in bacterial pathogenesis, remains largely unknown. Through the use of an L. interrogans htpG transposon insertion mutant, this study demonstrates that L. interrogans HtpG is essential for virulence in the hamster model of acute leptospirosis. Complementation of the htpG mutant completely restored virulence. Surprisingly, the htpG mutant did not appear to show sensitivity to heat or oxidative stress, phenotypes common in htpG mutants in other bacterial species. Furthermore, the mutant did not show increased sensitivity to serum complement, reduced survival within macrophages, or altered protein or lipopolysaccharide expression. The underlying cause for attenuation thus remains unknown, but HtpG is a novel leptospiral virulence factor and one of only a very small number identified to date.

Too old to be included: age diversity statements foster diversity yet fall short on inclusion.

Older employees often face discrimination and exclusion from work teams. In two scenario studies, we tested the impact of age diversity statements on the representation and inclusion of older employees in teams. In Study 1 (N = 304), participants had to create a team and were either exposed to a diversity statement or not before selecting two teammates from a list of four differing in age and gender. Then, we measured participants' inclusive behavioral intentions towards a new, older member joining this team. Age diversity statements increased the representation but not the inclusion of older individuals in teams. In Study 2 (N = 518), we further manipulated the content of the statement (diversity or diversity and inclusion) and the organizational motive (reputation or change). We replicated the effects of diversity statements on representation. Moreover, statements also increased certain inclusive behaviors, but only when they targeted diversity and inclusion and reflected an organizational commitment to change. Taken together, these results suggest that age diversity statements foster diversity, yet fail to systematically increase inclusion.

A systematic survey of environmental DNA in Palau's lakes and waterfalls reveals an increase in Leptospira levels after flooding.

Objective: Leptospirosis is an important bacterial zoonosis which is widespread in tropical and subtropical islands and influences human and animal health which has secondary economic effects. Although leptospirosis is endemic in Palau, an Oceanian Pacific Island country, few systematic surveys of potential risk factors for Leptospira infection, such as weather and host animals, have been conducted in the natural environment. We used environmental DNA metabarcoding to assess the distribution, species diversity, and abundance of pathogenic Leptospira in this endemic region to investigate the potential environmental risks. Methods: Forty-two paired water samples, representing fine and rainy weather conditions, were collected from four representative waterfalls and lakes on Babeldaob Island, the largest island in Palau. High-throughput sequencing analysis was conducted for polymerase chain reaction products of leptospiral 16S rRNA and vertebrate animal mitochondrial 12S rRNA genes. Results: We revealed greater Leptospira diversity and abundance in samples collected after continuous rain, particularly in the presence of flooding, compared with samples collected under typhoon, monsoon, or fine weather conditions. From same samples, six mammalian species including cats (Felis catus), mice (Mus musculus), Yap flying fox (Pteropus yapensis), rats (Rattus spp.), and pigs (Sus scrofa) were repeatedly detected. These may be candidates of host animals of Leptospira in Palau; however, their detection was not clearly correlated with that of Leptospira. Conclusion: We repeatedly detected several species of pathogenic Leptospira from water samples of a wide region of Babeldaob Island. We confirmed that Leptospira contamination in freshwater environments increased under rainy conditions, particularly in the presence of flooding. This information could be used to improve public health control measures in this region.

Enhancing the nitric oxide inhibitory activity using a combination of plant essential oils and mixture design approach.

The synergistic effects of essential oils (EOs) from three aromatic plant species, Foeniculum vulgare subsp. piperitum (C.Presl) Bég. (FV), Origanum heracleoticum L. (OH) and Lavandula austroapennina N.G.Passal., Tundis & Upson. (LA), were evaluated for their inhibitory properties on nitric oxide production in RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS). We utilized a Design of Experiments (DoE) methodology to optimize a formulation by combining three Essential Oils (EOs), while simultaneously taking into account two response variables, maximization of NO inhibition with minimum cytotoxicity. The optimal blend of components was predicted, and the statistical outcome's efficacy was experimentally verified. The combination corresponding to 87.7 % FV, 12.3 % LA and 0.0 % OH showed high inhibitory effect (76.3 %) with negligible cytotoxicity (4.5 %). This research provides new information on the interactions among fennel, oregano and lavender essential oils and shows how they can synergistically inhibit in vitro LPS-induced NO production.

Diagnostic Challenges in Hemophagocytic Lymphohistiocytosis, a Rare, Potentially Fatal Disease: Two Case Studies.

Hemophagocytic lymphohistiocytosis (HLH) is a rare, and potentially fatal, syndrome, characterized by immune system dysregulation, with excessive activation of the macrophages and cytotoxic T cells. It can be classified into primary (genetic) and secondary (acquired) forms. HLH presents with fever, hepatosplenomegaly, cytopenia, and hyperferritinemia, with involvement of various organs. The initial symptoms of HLH are non-specific, but as, if untreated, it can progress rapidly to multiorgan failure, timely diagnosis is essential. We present here two cases of HLH in infants that illustrate the importance of early diagnosis and appropriate treatment, along with a short review of HLH.

Pathological fracture induced by Halicephalobus gingivalis (Nematoda: Rhabditida) in a horse limb.

Halicephalobus gingivalis is a free-living nematode that occasionally causes infections in horses. We report a rare case of limb fracture of horse caused by infection with H. gingivalis. An 8-year-old mare was referred to the Veterinary Hospital of the Federal University of Lavras with claudication grade 5 of the right hind limb, that had been started 3 months ago. The patient had aseptic arthritis in the tarsal joint and edema that extended to the quartile. The radiographic examination showed punctate osteolysis with exacerbation of bone trabeculation along the calcaneus, talus, proximal epiphysis of the third metatarsal and distal epiphysis of the tibia. Treatment for arthritis was initiated, and the animal showed a slight improvement in limb function. However, 21 days after hospitalization, due to a comminuted fracture of the tibia, it was euthanized. At necropsy, yellowish masses were found from the metatarsal to the tibia, and around the tarsal bones and joint. Similar masses were also found in the left kidney. Numerous nematodes compatible with H. gingivalis were identified. This is the first description of a pathological fracture caused by H. gingivalis infection in an equine limb.

Inflammasome activation via intracellular NLRs triggered by bacterial infection.

Members of the nucleotide-binding, oligomerization domain (NOD)-like receptor (NLR) proteins assemble into a multiprotein platform, known as the inflammasome, to induce caspase-1 activation followed by the subsequent secretion of IL-1ß and IL-18. In this review, we focus on the role of NLRs in inflammasome activation as part of the host defence against bacterial pathogens. One of activators of the NLRC4 inflammasome is bacterial flagellin secreted through type III or IV secretion systems, which are important for the pathogenicity of many Gram-negative bacteria. The NLRP3 inflammasome is mainly activated by a large number of bacterial pore-forming toxins. Despite our knowledge of inflammasome activation upon bacterial infection, the function of antibacterial defence under in vivo conditions remains to be elucidated. Further understanding of NLR function should provide new insights into the mechanisms of host pro-inflammatory responses and the pathogenesis of bacterial infections.

Purification and use of E. coli peptide deformylase for peptide deprotection in chemoenzymatic peptide synthesis.

Peptide deformylases (PDFs) catalyze the removal of the formyl group from the N-terminal methionine residue in nascent polypeptide chains in prokaryotes. Its deformylation activity makes PDF an attractive candidate for the biocatalytic deprotection of formylated peptides that are used in chemoenzymatic peptide synthesis. For this application it is essential to use PDF preparations that are free of contamination by peptidases that can cleave internal peptide bonds. Therefore, different purification methods were attempted and an industrially applicable purification procedure was developed based on a single anion-exchange chromatography step of an engineered PDF variant that was equipped with an anionic octaglutamate tag. The deformylation activity and stability of the engineered enzyme were similar to those of the wild-type PDF. This purification method furnished a PDF preparation with a 1500-fold decreased level of contamination by amidases and peptidases as compared to cell-free extract. It was shown that the enzyme could be used for deprotection of a formylated dipeptide that was prepared by thermolysin-mediated coupling.

Degradation of p0071 and p120-catenin during adherens junction disassembly by Leptospira interrogans.

Leptospira interrogans disseminates hematogenously to reach the target organs by disrupting epithelial adherens junctions (AJs), thus causing leptospirosis, which is a globally neglected zoonotic disease. L. interrogans induces E-cadherin (E-cad) endocytosis and cytoskeletal rearrangement during AJ disassembly, but the detailed mechanism remains unknown. Elucidation of AJ disassembly mechanisms will guide new approaches to developing vaccines and diagnostic methods. In this study, we combine proteomic and imaging analysis with chemical inhibition studies to demonstrate that disrupting the AJs of renal proximal tubule epithelial cells involves the degradation of two armadillo repeat-containing proteins, p0071 and p120-catenin, that stabilize E-cad at the plasma membrane. Combining proteasomal and lysosomal inhibitors substantially prevented p120-catenin degradation, and monolayer integrity destruction without preventing p0071 proteolysis. In contrast, the pan-caspase inhibitor Z-VAD-FMK inhibited p0071 proteolysis and displacement of both armadillo repeat-containing proteins from the cell-cell junctions. Our results show that L. interrogans induces p120-catenin and p0071 degradation, which mutually regulates E-cad stability by co-opting multiple cellular degradation pathways. This strategy may allow L. interrogans to disassemble AJs and disseminate through the body efficiently.

Predictors of negative outcomes in hospitalized patients with SARS­CoV­2 pneumonia: A retrospective study.

The coronavirus disease 2019 (COVID-19) pandemic posed a serious threat to human health worldwide after the first case was identified in December 2019. Specific therapeutic options for COVID-19 are lacking; thus, the treatment of patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is complex in clinical practice. Despite the development of treatment options and methods to limit the spread of SARS-CoV-2, certain patients experience critical illness and numerous deaths have occurred. Notably, treatment of this disease is complex due to the evolution of viral mutations and variants with different rates of infection. Moreover, specific patient characteristics may be associated with rapid disease progression and poor outcomes. Thus, the present study aimed to identify the specific characteristics of patients who developed poor outcomes, including clinical manifestations, blood samples (blood cell count and coagulation tests) at hospital admission and comorbidities. The present study included a total of 1,813 patients hospitalized with pneumonia and SARS-CoV-2 infection, and mortality rates associated with each patient characteristic were calculated. The characteristics associated with the highest risk of mortality were as follows: Age >90 years (OR, 105; 95% CI, 17.70-2,023.00); oxygen saturation at the time of hospital admission <89% in room air (OR, 14.3; 95% CI, 7.54-30.7), admission to the Intensive Care Unit (OR, 39.4; 95% CI, 27.7-57.0); and a neutrophil/lymphocyte ratio of 8.76-54.2 (OR, 14; 95% CI, 7.62-29.0). Treatment of patients with SARS-CoV-2 pneumonia represents a challenge for the healthcare system, but there are a number of predictors for poor patient outcomes that could be identified at the time of hospital admission.