Engineered Chlorella vulgaris improves bioethanol production and promises prebiotic application.

Microalgal biomass for biofuel production, integration into functional food, and feed supplementation has generated substantial interest worldwide due to its high growth rate, non-competitiveness for agronomic land, ease of cultivation in containments, and presence of several bioactive molecules. In this study, genetic engineering tools were employed to develop transgenic lines of freshwater microalga Chlorella vulgaris with a higher starch content, by up-regulating ADP-glucose pyrophosphorylase (AGPase), which is a rate-limiting enzyme in starch biosynthesis. Expression of the Escherichia coli glgC (AGPase homolog) gene in C. vulgaris led to an increase in total carbohydrate content up to 45.1% (dry cell weight, DCW) in the transgenic line as compared to 34.2% (DCW) in the untransformed control. The starch content improved up to 16% (DCW) in the transgenic alga compared to 10% (DCW) in the control. However, the content of total lipid, carotenoid, and chlorophyll decreased differentially in the transgenic lines. The carbohydrate-rich biomass from the transgenic algal line was used to produce bioethanol via yeast fermentation, which resulted in a higher ethanol yield of 82.82 mg/L as compared to 54.41 mg/L from the untransformed control. The in vitro digestibility of the transgenic algal starch revealed a resistant starch content of up to 7% of total starch. Faster growth of four probiotic bacterial species along with a lowering of the pH of the growth medium indicated transgenic alga to exert a positive prebiotic effect. Taken together, the study documents the utilization of genetically engineered C. vulgaris with enriched carbohydrates as bioethanol feedstock and functional food ingredients.

Ancestral TALE homeobox protein transcription factor regulates actin dynamics and cellular activities of protozoan parasite Entamoeba invadens.

Entamoeba histolytica causes invasive amoebiasis, an important neglected tropical disease with a significant global health impact. The pathogenicity and survival of E. histolytica and its reptilian equivalent, Entamoeba invadens, relies on its ability to exhibit efficient motility, evade host immune responses, and exploit host resources, all of which are governed by the actin cytoskeleton remodeling. Our study demonstrates the early origin and the regulatory role of TALE homeobox protein EiHbox1 in actin-related cellular processes. Several genes involved in different biological pathways, including actin dynamics are differentially expressed in EiHbox1 silenced cells. EiHbox1 silenced parasites showed disrupted F-actin organization and loss of cellular polarity. EiHbox1's presence in the anterior region of migrating cells further suggests its involvement in maintaining cellular polarity. Loss of polarized morphology of EiHbox1 silenced parasites leads to altered motility from fast, directionally persistent, and highly chemotactic to slow, random, and less chemotactic, which subsequently leads to defective aggregation during encystation. EiHbox1 knockdown also resulted in a significant reduction in phagocytic capacity and poor capping response. These findings highlight the importance of EiHbox1 of E. invadens in governing cellular processes crucial for their survival, pathogenicity, and evasion of the host immune system.

Three-amino acid loop extension homeodomain proteins regulate stress responses and encystation in Entamoeba.

It is interesting to identify factors involved in the regulation of the encystation of Entamoeba histolytica that differentiate trophozoites into cysts. Evolutionarily conserved three amino acid loop extension (TALE) homeodomain proteins act as transcription factors and execute a variety of functions that are essential for life. A TALE homeodomain (EhHbox) protein-encoding gene has been identified in E. histolytica (Eh) that is highly upregulated during heat shock, glucose, and serum starvation. Its ortholog, EiHbox1, a putative homeobox protein in E. invadens (Ei), is also highly upregulated during the early hours of encystation, glucose starvation, and heat shock. They belong to the PBX family of TALE homeobox proteins and have conserved residues in the homeodomain that are essential for DNA binding. Both are localized in the nucleus during encystation and under different stress conditions. The electrophoretic mobility shift assay confirmed that the recombinant GST-EhHbox binds to the reported TGACAG and TGATTGAT motifs. Down-regulation of EiHbox1 by gene silencing reduced Chitin synthase, Jacob, and increased Jessie gene expression, resulting in defective cysts and decreased encystation efficiency and viability. Overall, our results suggest that the TALE homeobox family has been conserved during evolution and acts as a transcription factor to control the differentiation of Entamoeba by regulating the key encystation-induced genes.

A Comparison of Microwave Ablation and Cryoablation for the Treatment of Renal Cell Carcinoma: A Systematic Literature Review and Meta-analysis.

OBJECTIVE: To conduct a systematic review and meta-analysis comparing microwave ablation (MWA) and cryoablation for renal cell carcinoma (RCC). METHODS: The systematic search was performed in MEDLINE, Embase, and Cochrane databases. Studies published in English from January 2006 to February 2022 that assessed adults with primary RCC who received MWA or cryoablation were included. Study arms from RCTs, comparative observational, and single-arm studies were eligible. The outcomes included local tumor recurrence (LTR), overall survival, disease-free survival, overall/major complications, procedure/ablation time, 1- to 3-month primary technique efficacy, and technical success. Single-arm meta-analyses were performed using the random effects model. Sensitivity analyses excluding low-quality studies assessed using the MINORs scale were performed. Univariable and multivariable examined the effects of prognostic factors. RESULTS: Baseline characteristics were similar between groups and mean tumor size for MWA and cryoablation were 2.74 and 2.69 cm. Single-arm meta-analyses were similar for LTR and secondary outcomes between cryoablation and MWA. Ablation time was significantly shorter with MWA than with cryoablation (meta-regression weighted mean difference 24.55 minutes, 95% confidence interval -31.71, -17.38, P < .0001). One-year LTR was significantly lower with MWA than cryoablation (odds ratio 0.33, 95% confidence interval 0.10-0.93, P = .04). There were no significant differences for other outcomes. CONCLUSION: MWA provides significantly improved 1-year LTR and ablation time compared with cryoablation for patients with RCC. Other outcomes appeared similar or favorable for MWA; however, results were not statistically significant. MWA of primary RCC is as safe and effective as cryoablation, which should be confirmed with future comparative studies.

Stereotactic body radiation therapy and thermal ablation for treatment of NSCLC: A systematic literature review and meta-analysis.

RATIONALE: Stereotactic body radiation therapy (SBRT) is the standard of care for inoperable early stage non-small cell lung cancer (NSCLC). Use of image guided thermal ablation (IGTA; including microwave ablation [MWA] and radiofrequency ablation [RFA]) has increased in NSCLC, however there are no studies comparing all three. OBJECTIVE: To compare the efficacy of IGTA (including MWA and RFA) and SBRT for the treatment of NSCLC. METHODS: Published literature databases were systematically searched for studies assessing MWA, RFA, or SBRT. Local tumor progression (LTP), disease-free survival (DFS), and overall survival (OS) were assessed with single-arm pooled analyses and meta-regressions in NSCLC patients and a stage IA subgroup. Study quality was assessed with a modified methodological index for non-randomized studies (MINORS) tool. RESULTS: Forty IGTA study-arms (2,691 patients) and 215 SBRT study-arms (54,789 patients) were identified. LTP was lowest after SBRT at one and two years in single-arm pooled analyses (4% and 9% vs. 11% and 18%) and at one year in meta-regressions when compared to IGTA (OR = 0.2, 95%CI = 0.07-0.63). MWA patients had the highest DFS of all treatments in single-arm pooled analyses. In meta-regressions at two and three-years, DFS was significantly lower for RFA compared to MWA (OR = 0.26, 95%CI = 0.12-0.58; OR = 0.33, 95%CI = 0.16-0.66, respectively). OS was similar across modalities, timepoints, and analyses. Older age, male patients, larger tumors, retrospective studies, and non-Asian study region were also predictors of worse clinical outcomes. In high-quality studies (MINORS score ≥ 7), MWA patients had better clinical outcomes than the overall analysis. Stage IA MWA patients had lower LTP, higher OS, and generally lower DFS, compared to the main analysis of all NSCLC patients. CONCLUSIONS: NSCLC patients had comparable outcomes after SBRT and MWA, which were better than those with RFA.

Novel Insights into the Wattle and Daub Model of Entamoeba Cyst Wall Formation and the Importance of Actin Cytoskeleton.

The "Wattle and Daub" model of cyst wall formation in Entamoeba invadens has been used to explain encystment in Entamoeba histolytica, the causal agent of amoebiasis, and this process could be a potential target for new antiamoebic drugs. In this study, we studied the morphological stages of chitin wall formation in E. invadens in more detail using fluorescent chitin-binding dyes and the immunolocalization of cyst wall proteins. It was found that chitin deposition was mainly initiated on the cell surface at a specific point or at different points at the same time. The cystic wall grew outward and gradually covered the entire surface of the cyst over time, following the model of Wattle and Daub. The onset of chitin deposition was guided by the localization of chitin synthase 1 to the plasma membrane, occurring on the basis of the Jacob lectin in the cell membrane. During encystation, F-actin was reorganized into the cortical region within the early stages of encystation and remained intact until the completion of the chitin wall. The disruption of actin polymerization in the cortical region inhibited proper wall formation, producing wall-less cysts or cysts with defective chitin walls, indicating the importance of the cortical actin cytoskeleton for proper cyst wall formation.

Incremental Healthcare Cost Implications of Retreatment Following Ureteroscopy or Percutaneous Nephrolithotomy for Upper Urinary Tract Stones: A Population-Based Study of Commercially-Insured US Adults.

Purpose: This study describes the incremental healthcare costs associated with retreatment among adults undergoing ureteroscopy (URS) or percutaneous nephrolithotomy (PCNL) for upper urinary tract stones (UUTS). Patients and Methods: The IBM® MarketScan® Commercial Database was used to identify adults aged 18-64 years with UUTS treated with URS or PCNL between January 2010 and December 2019. Patients had 12 months of continuous insurance coverage before (baseline) and after (follow-up) the first (index) procedure. The primary outcome was total all-cause healthcare costs measured over the 365-day follow-up period, not inclusive of index costs. Generalized linear models were used to estimate the incremental costs associated with retreatment within 90 (early) or 91-365 days post-index (later) relative no retreatment. The models adjusted for demographics, comorbidities, stone(s) location, treatment setting, procedural characteristics (eg, 1-step vs 2-step PCNL) and index year. Results: Approximately 23% (27,402/119,800) of URS patients were retreated (82% had early retreatments). The adjusted mean total cost was $10,478 (95% CI: $10,281-$10,675) for patients with no retreatment, $25,476 (95% CI: $24,947-$26,004) for early retreatment ($14,998 incremental increase, p<0.01), and $32,868 [95% CI: $31,887-$33,850] for later retreatment ($22,391 incremental increase, p<0.01). Approximately 36% (1957/5516) of PCNL patients were retreated (78% had early retreatments). The adjusted mean total cost was $13,446 (95% CI: $12,659-$14,273) for patients with no retreatment, $37,036 [95% CI: $34,926-$39,145]) for early retreatment ($23,570 incremental increase, p<0.01), and $35,359 (95% CI: $32,234-$38,484) for later retreatment ($21,893 incremental increase, p<0.01). Conclusion: Retreatment during the first year following URS or PCNL was needed in 23% and 36% of patients, respectively, and was associated with an economic burden of up to $23,500 per patient. The high rate of retreatment and associated costs demonstrate there is an unmet need to improve mid- to long-term results in URS and PCNL.

Stress-responsive AMP Kinase like protein regulates encystation of Entamoeba invadens.

Starvation is always accompanied by an increase in the ratio of AMP/ATP followed by activation of AMPK. It is one of the sensors for cellular energy status and is highly conserved across various species. Its role in the stage differentiation process of protozoan species like Giardia, Plasmodium, Trypanosome, and Toxoplasma has been reported. Since Entamoeba undergoes encystation in glucose-starved conditions; it intrigued us to investigate the existence and role of AMPK during the differentiation of trophozoites to the cyst. By employing in silico approaches, we have identified an AMPK homologue which is denominated here as EiAMPK (AMPK-like protein in Entamoeba invadens). Sequence and structural analysis indicate that EiAMPK is sequentially and structurally similar to the AMPK alpha subunit of other organisms. The recombinant form of EiAMPK was functionally active and in accordance, its activity was inhibited by an AMPK-specific inhibitor (eg. Compound C). The increased expression of EiAMPK during different stresses indicated that EiAMPK is a stress-responsive gene. To further investigate, whether EiAMPK has any role in encystation, we employed RNAi-mediated gene silencing that demonstrated its active involvement in encystation. It is known that Entamoeba maintains a flow of glucose from the glycolytic pathway to chitin synthesis for cyst wall formation during encystation. It is conceivable that EiAMPK might have a command over such glucose metabolism. As anticipated, the chitin synthesis was found greatly inhibited in both EiAMPK knockdown and Compound C treated cells, indicating that EiAMPK regulates the cyst wall chitin synthesis.

Clinical profile of dermatitis neglecta with special emphasis on psychiatric comorbidities: A case series of 22 patients from Eastern India.

Dermatitis neglecta (DN), first described by Poskitt et al. in 1995, is a dermatosis resulting from insufficient frictional cleansing of the skin. Lesions result from self-neglect and may have several underlying etiologies, including psychiatric conditions. There is a dearth of any formal study or large case series on this subject. Moreover, the psychological aspects of DN mostly remain unexplored. We report here the clinical profile and psychiatric comorbidities of a group of patients with DN from eastern India. Sporadic cases of underlying psychological problems like cognitive deficit and executive dysfunction, schizophrenia, severe anxiety disorder, and depression have been reported in the existing literature. Associated psychiatric disorder was present in 72.7% of the patients of this case series that include several underlying psychiatric disorders like depression, schizophrenia, somatic symptom disorder, social anxiety disorder, intellectual disability, bipolar disorder, Alzheimer's disease, and alcohol dependence. In this article we would like to emphasize that DN is frequently associated with psychiatric disorder and management of the underlying psychiatric condition with psychotropic medications; psychotherapy is of immense importance.

Development of marker-free transgenic pigeon pea (Cajanus cajan) expressing a pod borer insecticidal protein.

Pigeon pea, a grain legume of the semiarid tropics, is a rich source of high-quality protein. The productivity of this pulse is seriously affected by lepidopteron insect pests. To generate a sustainable insect-resistant plant, synthetically prepared bioactive key constituents of a crystal protein (Syn Cry1Ab) of Bacillus thuringiensis were expressed in pigeon pea under the guidance of a tissue-specific promoter of the RuBP carboxylase/oxygenase small subunit (rbcS) gene. Regenerated transgenic plants with the cry1Ab expression cassette (cry1Ab-lox-bar-lox) showed the optimum insect motility rate (90%) in an in vitro insect bioassay with second instar larvae, signifying the insecticidal potency of Syn Cry1Ab. In parallel, another plant line was also generated with a chimaeric vector harbouring a cre recombinase gene under the control of the CaMV 2 × 35S promoter. Crossing between T1 plants with a single insertion of cry1Ab-lox-bar-lox T-DNA and T1 plants with moderate expression of a cre gene with a linked hygromycin resistance (hptII) gene was performed to exclude the bialaphos resistance (bar) marker gene. Excision of the bar gene was achieved in T1F1 hybrids, with up to 35.71% recombination frequency. Insect-resistant pigeon pea plants devoid of selectable marker genes (syn Cry1Ab- bar and cre-hptII) were established in a consecutive generation (T1F2) through genetic segregation.

Nucleotide sugar transporters of Entamoeba histolytica and Entamoeba invadens involved in chitin synthesis.

Chitin, a homopolymer of ß-(1,4) linked N-acetylglucosamine (GlcNAc), is a major component of cyst wall in the protozoan parasites Entamoeba histolytica (Eh) and Entamoeba invadens (Ei). The Entamoeba chitin synthase makes chitin at the vesicular membrane rather than the plasma membrane in fungi, even though the chemistry of chitin synthesis is most likely the same. However, the role of nucleotide sugar transporter(s) (NSTs) that are involved in chitin synthesis in Entamoeba are not yet established. In this study, we have identified the putative UDP-GlcNAc transporter (EiNst5) of Ei by BLASTP analysis using the amino acid sequence of EhNst3, the UDP-GlcNAc transporter of Eh. Heterologous expression of both EhNst3 and EiNst5 was found to complement the function of Yea4p (UDP-GlcNAc transporter of S. cerevisiae) in YEA4 null mutant and increased the cell wall chitin content. Like Yea4p in S. cerevisiae, Myc-epitope tagged EhNst3 and EiNst5 were localized to the endoplasmic reticulum in Δyea4 cells. The EiNST5 transcript was up-regulated during the in vitro encystation and oxidative stress in E. invadens. Similar up-regulation was also seen for EhNST3 under oxidative stress in E. histolytica. Down-regulation of EiNst5 expression using gene-specific dsRNA significantly reduced cyst formation during in vitro encystation in E. invadens. Our observations suggest for the first time the involvement of EhNst3 and EiNst5 in chitin synthesis and so in encystation of Entamoeba.

Association of tumor location with economic outcomes and air leak complications in thoracic lobectomies: results from a national hospital billing dataset.

Purpose: To assess whether tumor location during thoracic lobectomies affects economic outcomes or air leak complications. Patients and methods: Retrospective, observational study using Premier Healthcare Database. The study included patients aged ≥18 years who underwent elective inpatient thoracic lobectomy for lung cancer between 2012 and 2014 (first qualifying=index admission). Three mutually exclusive tumor location groups were formed: upper lobe, middle lobe, and lower lobe. Primary outcomes were index admission's length of stay (LOS), total hospital costs, and operating room time; in-hospital air leak complications (composite of air leak/pneumothorax) served as an exploratory outcome. Multivariable models were used to examine the association between tumor location and the study outcomes, accounting for covariates and hospital-level clustering. Results: 8,750 thoracic lobectomies were identified: upper lobe (n=5,284), middle lobe (n=512), and lower lobe (n=2,954). Compared with the upper lobe, the middle and lower lobe groups had statistically significant (p<0.05): shorter adjusted LOS (7.0 days upper vs 5.8 days middle, 6.6 days lower), lower adjusted mean total hospital costs ($26,177 upper vs $23,109 middle, $24,557 lower), and lower adjusted odds of air leak complications (odds ratio middle vs upper=0.81, 95% CI=0.74-0.89; odds ratio lower vs upper=0.60, 95% CI=0.46-0.78). Findings were similar but varied in statistical significance when stratified by open and video-assisted thoracoscopic surgery approach. Conclusion: Among patients undergoing elective thoracic lobectomy for lung cancer in real-world clinical practice, upper lobe tumors were significantly associated with increased in-hospital resource use and air leak complications as compared with lower or middle lobe tumors.