Transcriptome profiling of Plasmodium vivax in Saimiri monkeys identifies potential ligands for invasion.

Unlike the case in Asia and Latin America, Plasmodium vivax infections are rare in sub-Saharan Africa due to the absence of the Duffy blood group antigen (Duffy antigen), the only known erythrocyte receptor for the P. vivax merozoite invasion ligand, Duffy binding protein 1 (DBP1). However, P. vivax infections have been documented in Duffy-negative individuals throughout Africa, suggesting that P. vivax may use ligands other than DBP1 to invade Duffy-negative erythrocytes through other receptors. To identify potential P. vivax ligands, we compared parasite gene expression in Saimiri and Aotus monkey erythrocytes infected with P. vivax Salvador I (Sal I). DBP1 binds Aotus but does not bind to Saimiri erythrocytes; thus, P. vivax Sal I must invade Saimiri erythrocytes independent of DBP1. Comparing RNA sequencing (RNAseq) data for late-stage infections in Saimiri and Aotus erythrocytes when invasion ligands are expressed, we identified genes that belong to tryptophan-rich antigen and merozoite surface protein 3 (MSP3) families that were more abundantly expressed in Saimiri infections compared with Aotus infections. These genes may encode potential ligands responsible for P. vivax infections of Duffy-negative Africans.

Activity of Plasmodium vivax promoter elements in Plasmodium knowlesi, and a centromere-containing plasmid that expresses NanoLuc throughout the parasite life cycle.

BACKGROUND: Plasmodium knowlesi is now the major cause of human malaria in Malaysia, complicating malaria control efforts that must attend to the elimination of multiple Plasmodium species. Recent advances in the cultivation of P. knowlesi erythrocytic-stage parasites in vitro, transformation with exogenous DNA, and infection of mosquitoes with gametocytes from culture have opened up studies of this pathogen without the need for resource-intensive and costly non-human primate (NHP) models. For further understanding and development of methods for parasite transformation in malaria research, this study examined the activity of various trans-species transcriptional control sequences and the influence of Plasmodium vivax centromeric (pvcen) repeats in plasmid-transfected P. knowlesi parasites. METHODS: In vitro cultivated P. knowlesi parasites were transfected with plasmid constructs that incorporated Plasmodium vivax or Plasmodium falciparum 5' UTRs driving the expression of bioluminescence markers (firefly luciferase or Nanoluc). Promoter activities were assessed by bioluminescence, and parasites transformed with human resistant allele dihydrofolate reductase-expressing plasmids were selected using antifolates. The stability of transformants carrying pvcen-stabilized episomes was assessed by bioluminescence over a complete parasite life cycle through a rhesus macaque monkey, mosquitoes, and a second rhesus monkey. RESULTS: Luciferase expression assessments show that certain P. vivax promoter regions, not functional in the more evolutionarily-distant P. falciparum, can drive transgene expression in P. knowlesi. Further, pvcen repeats may improve the stability of episomal plasmids in P. knowlesi and support detection of NanoLuc-expressing elements over the full parasite life cycle from rhesus macaque monkeys to Anopheles dirus mosquitoes and back again to monkeys. In assays of drug responses to chloroquine, G418 and WR9910, anti-malarial half-inhibitory concentration (IC50) values of blood stages measured by NanoLuc activity proved comparable to IC50 values measured by the standard SYBR Green method. CONCLUSION: All three P. vivax promoters tested in this study functioned in P. knowlesi, whereas two of the three were inactive in P. falciparum. NanoLuc-expressing, centromere-stabilized plasmids may support high-throughput screenings of P. knowlesi for new anti-malarial agents, including compounds that can block the development of mosquito- and/or liver-stage parasites.

Artemisinin resistance phenotypes and K13 inheritance in a Plasmodium falciparum cross and Aotus model.

Concerns about malaria parasite resistance to treatment with artemisinin drugs (ARTs) have grown with findings of prolonged parasite clearance t1/2s (>5 h) and their association with mutations in Plasmodium falciparum Kelch-propeller protein K13. Here, we describe a P. falciparum laboratory cross of K13 C580Y mutant with C580 wild-type parasites to investigate ART response phenotypes in vitro and in vivo. After genotyping >400 isolated progeny, we evaluated 20 recombinants in vitro: IC50 measurements of dihydroartemisinin were at similar low nanomolar levels for C580Y- and C580-type progeny (mean ratio, 1.00; 95% CI, 0.62-1.61), whereas, in a ring-stage survival assay, the C580Y-type progeny had 19.6-fold (95% CI, 9.76-39.2) higher average counts. In splenectomized Aotus monkeys treated with three daily doses of i.v. artesunate, t1/2 calculations by three different methods yielded mean differences of 0.01 h (95% CI, -3.66 to 3.67), 0.80 h (95% CI, -0.92 to 2.53), and 2.07 h (95% CI, 0.77-3.36) between C580Y and C580 infections. Incidences of recrudescence were 57% in C580Y (4 of 7) versus 70% in C580 (7 of 10) infections (-13% difference; 95% CI, -58% to 35%). Allelic substitution of C580 in a C580Y-containing progeny clone (76H10) yielded a transformant (76H10C580Rev) that, in an infected monkey, recrudesced regularly 13 times over 500 d. Frequent recrudescences of ART-treated P. falciparum infections occur with or without K13 mutations and emphasize the need for improved partner drugs to effectively eliminate the parasites that persist through the ART component of combination therapy.

Predictors of catheter-free voiding after spinal cord injury.

CONTEXT: Despite a high prevalence of neurogenic bladder (NGB) in patients with spinal cord injury (SCI), clinicians are unable to predict long-term bladder outcomes due to variable phenotypes of bladder dysfunction. This study investigates if early bladder events, infections, and spinal cord injury characteristics during rehabilitation admission affect bladder outcomes one year after SCI. METHODS: This retrospective study included patients with SCI admitted to a tertiary rehabilitation center between 1 January 2016 and 1 January 2020. Data was collected on early bladder management, comorbidities, infections and injury characteristics; level of injury, American Spinal Injury Association Impairment Scale (AIS) classification, and International Standards for Neurological Classification of Spinal Cord Injury lower extremity motor score (LEMS). RESULTS: Seventy-two patients met inclusion criteria; 63% (45/72) patients had cervical SCI and 31% (22/72) were complete injuries. Twenty-two percent (16/72) did not use an internal catheter to empty the bladder, improving to 41% (29/72) at one year. On multivariate logistic regression accounting for age, sex, Charlson comorbidity index, LEMS, and infections during admission, higher LEMS (OR 1.104, 95%, CI 1.037-1.176, P = 0.002) associated with catheter-free voiding (CFV) at one year while male sex (OR 0.091, 95% CI 0.012-0.713, P = 0.0225), and non-urologic infections (OR 0.088, 95% CI 0.010-0.768, P = 0.0279) were negatively associated. CONCLUSIONS: Preserved LEMS early after SCI associates with CFV at one year while male sex and early non-urologic infections such as pneumonia are associated with persistent urinary retention. This can be used to counsel SCI patients on expected bladder recovery and outcomes.

Microwave ablation of the T1a small renal mass: expanding beyond 3 cm.

PURPOSE: To compare the oncological and renal function outcomes for patients receiving microwave ablation (MWA) in tumors < 3 and 3-4 cm. METHODS: Retrospective analysis of a prospectively maintained database identified patients with < 3 or 3-4 cm renal cancers undergoing MWA. Radiographic follow-up occurred at approximately 6 months post-procedure and annually thereafter. Serum creatinine and estimated glomerular filtration rate (eGFR) were calculated before and 6-months post-MWA. Local recurrence-free survival (LRFS) was estimated using the Kaplan-Meier method. Tumor size was evaluated as a prognostic factor using Cox proportional-hazards regression. Predictors for change in eGFR and chronic kidney disease (CKD) stage were modeled using linear and ordinal logistic regression. RESULTS: A total of 126 patients fit the inclusion criteria. Overall recurrences were 2/62 (3.2%) and 6/64 (9.4%) for < 3 versus 3-4 cm. Both recurrences in the < 3 cm group were local, 4/6 in the 3-4 cm group were local and 2/6 were metastatic without local progression. For < 3 versus 3-4 cm, cumulative LRFS at 36 months was 94.6% versus 91.4%. Tumor size was not a significant prognostic factor for LRFS. Renal function did not change significantly after MWA. Patient comorbidities and RENAL nephrometry score significantly affected change in CKD. CONCLUSION: With comparable oncological outcomes, complication rates, and renal function preservation, MWA is a promising management strategy for renal masses of 3-4 cm in select patients. Our findings suggest that current AUA guidelines, which recommend thermal ablation for tumors < 3 cm, may need review to include T1a tumors for MWA, regardless of size.

Cytoplasmic pH response to acid stress in individual cells of Escherichia coli and Bacillus subtilis observed by fluorescence ratio imaging microscopy.

The ability of Escherichia coli and Bacillus subtilis to regulate their cytoplasmic pH is well studied in cell suspensions but is poorly understood in individual adherent cells and biofilms. We observed the cytoplasmic pH of individual cells using ratiometric pHluorin. A standard curve equating the fluorescence ratio with pH was obtained by perfusion at a range of external pH 5.0 to 9.0, with uncouplers that collapse the transmembrane pH difference. Adherent cells were acid stressed by switching the perfusion medium from pH 7.5 to pH 5.5. The E. coli cytoplasmic pH fell to a value that varied among individual cells (range of pH 6.2 to 6.8), but a majority of cells recovered (to pH 7.0 to 7.5) within 2 min. In an E. coli biofilm, cells shifted from pH 7.5 to pH 5.5 failed to recover cytoplasmic pH. Following a smaller shift (from pH 7.5 to pH 6.0), most biofilm cells recovered fully, although the pH decreased further than that of isolated adherent cells, and recovery took longer (7 min or longer). Some biofilm cells began to recover pH and then failed, a response not seen in isolated cells. B. subtilis cells were acid shifted from pH 7.5 to pH 6.0. In B. subtilis, unlike the case with E. coli, cytoplasmic pH showed no "overshoot" but fell to a level that was maintained. This level of cytoplasmic pH post-acid shift varied among individual B. subtilis cells (range of pH, 7.0 to 7.7). Overall, the cytoplasmic pHs of individual bacteria show important variation in the acid stress response, including novel responses in biofilms.

Recurrent Anterior Urethral Stricture: Challenges and Solutions.

Recurrent male anterior urethral stricture disease is a complex surgical challenge that should be managed by reconstructive urologists with experience in stricture management. Diagnosis of recurrence requires both anatomic narrowing and patient symptoms identified on validated questionnaires, with limited role for intervention in asymptomatic treatment "failures". Endoscopic management has a very specific role in recurrence, and the choice of technique for urethroplasty depends on pre-operative urethrography and cystoscopy. Surgical success depends on addressing patient concerns, complete stricture excision, tissue quality optimization, and the use of multi-stage repair when indicated. Augmentation with genital skin flaps and/or grafts is often required, with buccal mucosa as the ideal graft source if local tissue is compromised. Salvage options including urinary diversion and perineal urethrostomy must also be considered in debilitated patients with severe disease or repeated treatment failures. Unique patient populations including patients with hypospadias and lichen sclerosis are among the highest risk for repeated recurrence and require special care in surgical technique, graft selection, and post-operative management.

Thermal ablation of the small renal mass: a critical analysis of current literature.

INTRODUCTION: Thermal ablation is increasingly utilized as a management strategy for small renal masses (<4 cm). Partial nephrectomy is recognized as the gold standard; thermal ablation has been reserved for older patients with comorbidities due to concern for local tumor recurrence. As long-term data regarding the safety and efficacy of ablative techniques accumulate with encouraging results, clinicians are widening the utility in select patient populations. This review summarizes the currently available technologies in terms of procedural differences, oncologic outcomes, renal function, and complication rates. EVIDENCE ACQUISITION: A structured literature review was conducted using PubMed and Web of Science, using the keywords: "renal cell carcinoma," "ablation techniques," "cryosurgery," "radiofrequency ablation," "microwave ablation," "outcomes assessment," "post-operative complication," and "hospital costs." Articles were reviewed to summarize oncologic outcomes, complications, and impact on renal function of cryoablation, radiofrequency ablation, and microwave ablation. EVIDENCE SYNTHESIS: Thermal ablation is a safe and effective management option for small renal masses in select patients, particularly in those with multiple tumors and/or those unable or unwilling to undergo more invasive surgery. Slightly higher rates of local recurrence rates (~1-10%) with thermal ablation are offset by lower complication rates and reduced morbidity, and equivalent or better renal function outcomes compared to surgery. CONCLUSIONS: The established modalities of cryo-, radiofrequency, and microwave ablation offer equivalent outcomes with similar complication rates; technique choice is primarily based on tumor characteristics and operator preference. Better quality evidence comparing thermal ablation with surgical nephron-sparing intervention is needed to make informed conclusions on efficacy.

Infection of mosquitoes from in vitro cultivated Plasmodium knowlesi H strain.

In vitro studies of sexual blood stages of the most fatal malaria species, Plasmodium falciparum, have revealed key processes by which gametocytes develop and transmit infection from humans to anopheline mosquitoes. However, most malaria cases outside sub-Saharan Africa are caused by other Plasmodium spp., frequently Plasmodium vivax and Plasmodium knowlesi, a zoonotic parasite of macaque monkeys. Gametocytes of P. vivax and P. knowlesi exhibit distinct morphology, faster development, and a shorter life span compared with gametocytes of P. falciparum, reflecting the evolutionary separation and biological differences of these species. Unlike P. falciparum, P. vivax cannot be cultivated in vitro, necessitating access to infected primates for laboratory studies. In contrast, P. knowlesi asexual stages have been successfully adapted to cultures in macaque and human red blood cells, but these stages have not been reported to produce gametocytes infective to mosquitoes. Here, we show that gametocyte production and sporadic, low-level mosquito infectivity of a P. knowlesi strain was not improved by application of a "crash" method commonly used to induce gametocytes in P. falciparum cultures. However, Percoll-gradient purified schizonts from this strain yielded highly synchronised populations that, in three of six experiments, produced infections at an average rate of 0.97-9.1 oocysts in Anopheles dirus mosquitoes. Oocyst counts were most abundant in mosquitoes that were fed from the synchronised cultures 36 h after schizont purification. Gametocytes in these cultures occurred at low prevalence and were difficult to observe. Transcription from orthologs of P. falciparum gametocyte-specific markers did not correlate with infectivity of the P. knowlesi parasites to mosquitoes. The ability to infect mosquitoes from in vitro-cultivated P. knowlesi will support research on the unique features of this emerging pathogen and facilitate comparative studies of transmission by the different human malarias.