Coronavirus 2019 disease: Are corticosteroids the key treatment? A retrospective case-control study in Brazil.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is the putative cause of coronavirus disease 2019 (COVID-19), a serious disease that has severely impacted the world. Although vaccines have been developed, it will take time to inoculate the global population. Current guidelines have focused on the treatment of severe cases in hospital settings; however, a void has been created regarding appropriate measures for those in the initial stage of COVID-19 and those experiencing moderate disease severity progressing to desaturation. We assessed clinical outcomes in patients with COVID-19 with pneumonia at initial presentation treated with corticosteroids. Methods: Data of 177 consecutive high-risk patients with COVID-19, monitored by telemedicine, were collected and analyzed. Of those, 68 patients were in the initial inflammatory phase of the disease without desaturation and received corticosteroids. The outcomes were evaluated after a follow up of 14 days. Four patients were immediately referred to the hospital because they had explicit desaturation at presentation. Results: After 14 days, all patients in the inflammatory phase at presentation who were treated with corticosteroids before desaturation were alive and without complications. However, of the four patients with desaturation, one died at the hospital. Conclusion: In this study, the use of corticosteroids during the initial pulmonary phase of COVID-19 before desaturation, in addition to daily monitoring of patients, prevented disease progression, decreased the risk of complications and incidence of hospitalization and death. However, additional studies with larger number of patients are needed to confirm these findings.

Leprosy detection rate in patients under immunosuppression for the treatment of dermatological, rheumatological, and gastroenterological diseases: a systematic review of the literature and meta-analysis.

BACKGROUND: Recently developed immunosuppressive drugs, especially TNF antagonists, may enhance the risk of granulomatous infections, including leprosy. We aimed to evaluate the leprosy detection rate in patients under immunosuppression due to rheumatological, dermatological and gastroenterological diseases. METHODS: We performed a systematic review of the literature by searching the PubMed, EMBASE, LILACS, Web of Science and Scielo databases through 2018. No date or language restrictions were applied. We included all articles that reported the occurrence of leprosy in patients under medication-induced immunosuppression. RESULTS: The search strategy resulted in 15,103 articles; finally, 20 articles were included, with 4 reporting longitudinal designs. The detection rate of leprosy ranged from 0.13 to 116.18 per 100,000 patients/year in the USA and Brazil, respectively. In the meta-analysis, the detection rate of cases of leprosy per 100,000 immunosuppressed patients with rheumatic diseases was 84 (detection rate = 0.00084; 95% CI = 0.0000-0.00266; I2 = 0%, p = 0.55). CONCLUSION: Our analysis showed that leprosy was relatively frequently detected in medication-induced immunosuppressed patients suffering from rheumatological diseases, and further studies are needed. The lack of an active search for leprosy in the included articles precluded more precise conclusions. TRIAL REGISTRATION: This review is registered in PROSPERO with the registry number CRD42018116275 .

Spontaneous expulsion of a submucosal uterine fibroid without embolization in a pre-menopausal woman.

Uterine leiomyomas are benign tumors that develop from smooth muscle tissue and are present in up to 77% of women in menacme. They are often asymptomatic but can cause pelvic pain, compression, abnormal uterine bleeding, and degeneration. We present the first case report of a perimenopausal woman who exhibited complete and spontaneous expulsion of uterine fibroids without embolization or use of medication. She complained of a mass extruding from the vaginal orifice associated with bleeding and pain for a couple of hours. The anatomopathological findings showed a myomatous lesion. Complete expulsion of a uterine fibroid is a rare condition that may be associated with profuse hemorrhage and can pose a risk to the patient. When it occurs during perimenopause, it can mimic several clinical conditions. Therefore, gynecologists must remain alert to make the correct diagnosis and treatment.

An alternative approach to vaginal dilation in patients with Meyer-Rokitanski-Küster-Hauser syndrome: two case reports.

Vaginal dilation, currently considered as the first-line therapy for vaginal aplasia in patients with Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome, is a safe and effective treatment that aims to create a functional neovagina. However, rigid vaginal dilators classically described in the literature usually cause physical discomfort and side effects that can lead to vaginal necrosis. Here, we present two cases of MRKH syndrome patients with vaginal agenesis whose main complaint was the inability to have sexual intercourse with their partners. Considering unavailability of acrylic dilators and previous studies reporting good responses with the use of silicone dilators in women with post-radiotherapy vaginal stenosis, the medical team and patients opted for creation of a neovagina through the daily use of silicone vaginal dilators. Patient 1 developed an 8-cm vagina after 6 months of treatment and had a satisfactory sex life with her partner. Patient 2 developed a 7-cm vagina and reported significant symptom improvement. None of the patients developed side effects after the treatment. The use of inexpensive and easily accessible silicone vaginal dilators may be an effective and noninvasive alternative with few side effects for women with vaginal agenesis, particularly in the developing countries.

Hydrogen-bond-based protein engineering for the acidic adaptation of Bacillus acidopullulyticus pullulanase.

Pullulanase is a starch-debranching enzyme that is generally employed to efficiently break down starch for the production of high-glucose syrup. Acidic adaptation of pullulanases is of special interest. In this study, we conducted protein engineering to improve the acidic adaptation of Bacillus acidopullulyticus pullulanase (BaPul) and used a hydrogen-bond-based approach to identify promising residues that may change the deprotonation constants (pKa) of the catalytic residues. A total of 19 amino acids were selected for mutation according to the crystal structure of BaPul. The pH optimum of the L627R mutant shifted from 5.0 to 4.0, and its relative activity at pH 4.0 was 117% that of the wide-type enzyme. The improved efficacy of the L627R mutant at pH 4.0 was confirmed by kinetic parameters and pKa prediction. Moreover, the L627R mutant exhibited increased tolerance against acid-mediated denaturation, and its maximum d-glucose content (97.4%) was obtained after 40 h incubation, which is shorter by 10 h compared with the time required by the wide-type enzyme to produce a comparable amount of the monosaccharide. The L627R mutant may be suitable for industrial application because its shortened reaction time translates to reduced energy consumption.

Improvement of enzymatic properties of Rhizopus oryzae α-amylase by site-saturation mutagenesis of histidine 286.

Optimal pH and ideal functioning temperature for fungal α-amylase can greatly contribute to improving enzyme efficiency in maltose-forming ability. This work aimed to improve the enzymatic properties of Rhizopus oryzae α-amylase by site-saturation mutagenesis of histidine 286. The biochemical properties of selected mutant enzymes were modified to increase their enzymatic efficiencies compared to their wild-type counterparts. For instance, the optimum temperature of mutants H286 L, H286I, H286S and H286 T was increased from 50 °C to 55 °C, while a similar increase was observed for H286 P from 50 °C to 60 °C. The optimum pH of mutants H286 L, H286I and H286D shifted from 5.5 to 5.0, and the optimum pH of mutant H286E shifted from 5.5 to 4.5. The results obtained showed that the mutant H286I showed a 1.5-fold increase in half-life at 55 °C and the mutant H286E showed a 6.43-fold increase in half-life at a pH of 4.5. Furthermore, the ability to form maltose from soluble starch for mutants H286 L and H286 M was significantly improved under the optimum conditions determined in the study. The catalytic mechanism responsible for improved maltose-forming ability was confirmed through molecular docking simulations with maltotriose among wild-type and mutant enzymes. The mutants with improved enzymatic properties that were attained in this work may help in future computer-aided directed evolution of fungal α-amylase.

Conservative treatment and follow-up of vaginal Gartner's duct cysts: a case series.

BACKGROUND: In women, during embryologic development, the paired Müllerian (paramesonephric) ducts fuse distally and develop into the uterus, cervix, and upper vagina. If the Wolffian ducts persist in vestigial form, they can lead to Gartner's cysts, mainly located in the right wall of the vagina. This is one of the few studies of Gartner's cysts with a series of consecutive cases over a long period of time who were exclusively subject to clinical observation. Although Gartner's cysts are found in approximately 0.1 to 0.2% of women, controversy exists regarding the course of action to be taken. CASE PRESENTATION: We describe the cases of four women who were 38-years old, 53-years old, 37-years old, and 49-years old at their first appointment and who were of mixed ethnicity, mixed ethnicity, black, and mixed ethnicity respectively. The follow-up of these patients ranged from 2 to 17 years. In these four cases the location of the cysts was the right wall of the vagina. Transvaginal ultrasound was the test of choice for diagnostic confirmation. In the cases presented in this study, the women were asymptomatic and chose to be observed clinically. CONCLUSIONS: This is the first study reporting long-term clinical observation of these lesions. This study shows that conservative treatment can be a safe option for asymptomatic patients with vaginal Gartner's duct cysts.

[Effect of N-terminal truncation of Bacillus acidopullulyticus pullulanase on enzyme properties and functions].

We constructed different N-terminal truncated variants based on Bacillus acidopullulyticus pullulanase 3D structure (PDB code 2WAN), and studied the effects of truncated mutation on soluble expression, enzymatic properties, and application in saccharification. Upon expression, the variants of X45 domain deletion existed as inclusion bodies, whereas deletion of CBM41 domain had an effective effect on soluble expression level. The variants that lack of CBM41 (M1), lack of X25 (M3), and lack both of CBM41 and X25 (M5) had the same optimal pH (5.0) and optimal temperature (60 degrees C) with the wild-type pullulanase (WT). The K(m) of M1 and M5 were 1.42 mg/mL and 1.85 mg/mL, respectively, 2.4- and 3.1-fold higher than that of the WT. k(cat)/K(m) value of M5 was 40% lower than that of the WT. Substrate specificity results show that the enzymes exhibited greater activity with the low-molecular-weight dextrin than with high-molecular-weight soluble starch. When pullulanases were added to the saccharification reaction system, the dextrose equivalent of the WT, M1, M3, and M5 were 93.6%, 94.7%, 94.5%, and93.1%, respectively. These results indicate that the deletion of CBM41 domain and/or X25 domain did not affect the practical application in starch saccharification process. Furthermore, low-molecular-weight variants facilitate the heterologous expression. Truncated variants may be more suitable for industrial production than the WT.

The Impact of Inventory Management on Stock-Outs of Essential Drugs in Sub-Saharan Africa: Secondary Analysis of a Field Experiment in Zambia.

OBJECTIVE: To characterize the impact of widespread inventory management policies on stock-outs of essential drugs in Zambia's health clinics and develop related recommendations. METHODS: Daily clinic storeroom stock levels of artemether-lumefantrine (AL) products in 2009-2010 were captured in 145 facilities through photography and manual transcription of paper forms, then used to determine historical stock-out levels and estimate demand patterns. Delivery lead-times and estimates of monthly facility accessibility were obtained through worker surveys. A simulation model was constructed and validated for predictive accuracy against historical stock-outs, then used to evaluate various changes potentially affecting product availability. FINDINGS: While almost no stock-outs of AL products were observed during Q4 2009 consistent with primary analysis, up to 30% of surveyed facilities stocked out of some AL product during Q1 2010 despite ample inventory being simultaneously available at the national warehouse. Simulation experiments closely reproduced these results and linked them to the use of average past monthly issues and failure to capture lead-time variability in current inventory control policies. Several inventory policy enhancements currently recommended by USAID | DELIVER were found to have limited impact on product availability. CONCLUSIONS: Inventory control policies widely recommended and used for distributing medicines in sub-Saharan Africa directly account for a substantial fraction of stock-outs observed in common situations involving demand seasonality and facility access interruptions. Developing central capabilities in peripheral demand forecasting and inventory control is critical. More rigorous independent peer-reviewed research on pharmaceutical supply chain management in low-income countries is needed.

Downsizing a pullulanase to a small molecule with improved soluble expression and secretion efficiency in Escherichia coli.

BACKGROUND: Significant challenges, including low expression and extracellular secretion of soluble protein, are encountered in expressing and purifying Bacillus acidopullulyticus pullulanase (BaPul) in Escherichia coli. METHODS: An N-terminal domain truncation was adopted to facilitate BaPul variant expression and/or secretion. RESULTS: BaPul possesses a complex modular architecture that consists of CBM41-X45a-X25-X45b-CBM48-GH13. The activities of M1 (ΔCBM41) and M5 (ΔCBM41ΔX25) variants were 2.9- and 2.4-fold that of wild-type (WT) enzyme, respectively. The enhanced expression of soluble protein is the main reason for these improved activities. PelB-M1 and PelB-M5 were transported to the periplasmic space, where PelB is part of the PelB-pET28a(+) construct, and PelB-M3 (ΔX25) and PelB-WT variants were largely retained in the cytoplasm. After fermentation, about 56.6 and 93.4 % of the total activity of PelB-M1 and PelB-M5 were transferred to the periplasm, respectively, followed by cell lysis and leakage of the partial enzyme into the extracellular medium. The optimal temperature and pH for purified preparations of M1, M3, and M5 were similar to those of the WT enzyme. In a starch saccharification reaction, the dextrose equivalents of M1, M3, and M5 proteins were 94.7, 94.5, and 93.1 %, respectively, which were also essentially identical to that of WT (93.6 %). CONCLUSION: The deletion of CBM41 and/or X25 domain did not affect the enzyme application, and the truncated variants were more highly expressed and secreted in E. coli. Thus, the truncated variants may be more suitable for industrial applications.

Protein engineering of Bacillus acidopullulyticus pullulanase for enhanced thermostability using in silico data driven rational design methods.

Thermostability has been considered as a requirement in the starch processing industry to maintain high catalytic activity of pullulanase under high temperatures. Four data driven rational design methods (B-FITTER, proline theory, PoPMuSiC-2.1, and sequence consensus approach) were adopted to identify the key residue potential links with thermostability, and 39 residues of Bacillus acidopullulyticus pullulanase were chosen as mutagenesis targets. Single mutagenesis followed by combined mutagenesis resulted in the best mutant E518I-S662R-Q706P, which exhibited an 11-fold half-life improvement at 60 °C and a 9.5 °C increase in Tm. The optimum temperature of the mutant increased from 60 to 65 °C. Fluorescence spectroscopy results demonstrated that the tertiary structure of the mutant enzyme was more compact than that of the wild-type (WT) enzyme. Structural change analysis revealed that the increase in thermostability was most probably caused by a combination of lower stability free-energy and higher hydrophobicity of E518I, more hydrogen bonds of S662R, and higher rigidity of Q706P compared with the WT. The findings demonstrated the effectiveness of combined data-driven rational design approaches in engineering an industrial enzyme to improve thermostability.

A case of swyer syndrome associated with advanced gonadal dysgerminoma involving long survival.

Swyer syndrome is caused by abnormal sex differentiation during the embryonic period, resulting in incomplete intrauterine masculinization and undifferentiated gonads. The current case report describes a patient with Swyer syndrome associated with stage 3 gonadal dysgerminoma who has survived for 23 years. At age 18, this patient sought assistance for primary amenorrhea from the Gynecological Services Department of the University of Brasília Hospital. A physical examination revealed that the patient was at Tanner stage 4 with respect to axillary hair, breasts, and pubic hair; she presented with a eutrophic vagina and a small cervix. She was treated with a combination of estrogens and progestogens to induce cycling. Approximately 4 years later, a complex tumor was found and resected; a histopathological analysis revealed that this tumor was a right adnexal dysgerminoma with peritoneal affection. The patient was also subjected to chemotherapy. Her follow-up has continued to the present time, with no signs of tumor recurrence. In conclusion, this report describes an extremely rare case in which Swyer syndrome was associated with ovarian dysgerminoma; relative to similar patients, the described patient has survived for an unusually prolonged time.

Integrative investigation on breast cancer in ER, PR and HER2-defined subgroups using mRNA and miRNA expression profiling.

Exploring the molecular difference among breast cancer subtypes is of crucial importance in understanding its heterogeneity and seeking its effective clinical treatment. For this, several layers of information including immunohistochemical markers and a variety of high-throughput genomics approaches have been intensively used. Here we have explored the intrinsic differences among breast cancer subgroups defined by immunohistochemical expression (IHC) of hormone receptors ER and PR as well as human epidermal growth factor receptor 2 (HER2) using the mRNA and miRNA expression profiles of 115 tumors. A core basal group was further defined by epidermal growth factor receptor and cytokeratin 5/6 IHC expression and compared to triple negative group. A set of differentially expressed genes including 1015 mRNAs and 69 miRNAs was found to distinguish tumor subtypes whose generality was demonstrated using two independent data sets. The network was explored for each subtype and biomass synthesis signaling was found to play an important role in the core basal subgroup. This study contributes to elucidating the intrinsic relations among breast cancer subgroups defined by ER, PR and HER2 expression via integrating mRNA and miRNA expression. The results can avail functional studies of breast cancer with translational potential for clinical use.

Soluble expression of pullulanase from Bacillus acidopullulyticus in Escherichia coli by tightly controlling basal expression.

Bacillus acidopullulyticus pullulanase (BaPul13A) is a widely used debranching enzyme in the starch industry. A few details have been reported on the heterologous expression of BaPul13A in Escherichia coli (E. coli). This study compares different E. coli expression systems to improve the soluble expression level of BaPul13A. When pET22b(+)/pET28a(+) was used as the expression vector, the soluble expression of BaPul13A can be achieved by tightly controlling basal expression, whereas pET-20b(+)/pGEX4T2 leads to insoluble inclusion bodies. An efficient process control strategy aimed at minimizing the formation of inclusion bodies and enhancing the production of pullulanase was developed by a step decrease of the temperature in a 5-L fermentor. The highest total enzyme activity of BaPul13A reached 1,156.32 U/mL. This work reveals that the T7 promoter with lac operator and lacI gene collectively contribute to the soluble expression of BaPul13A, whereas either a T7 promoter alone or combined with the lac operator and lacI gene results in poor solubility. Basal expression in the initial growth phase of the host significantly affects the solubility of BaPul13A in E. coli.