Expression and Purification of His-Tagged Variants of Human Hepatitis A Virus 3C Protease.

BACKGROUND: Protease 3C (3Cpro) is the only protease encoded in the human hepatitis A virus genome and is considered as a potential target for antiviral drugs due to its critical role in the viral life cycle. Additionally, 3Cpro has been identified as a potent inducer of ferroptosis, a newly described type of cell death. Therefore, studying the molecular mechanism of 3Cpro functioning can provide new insights into viral-host interaction and the biological role of ferroptosis. However, such studies require a reliable technique for producing the functionally active recombinant enzyme. OBJECTIVE: Here, we expressed different modified forms of 3Cpro with a hexahistidine tag on the N- or C-terminus to investigate the applicability of immobilized metal Ion affinity chromatography (IMAC) for producing 3Cpro. METHODS: We expressed the proteins in Escherichia coli and purified them using IMAC, followed by gel permeation chromatography. The enzymatic activity of the produced proteins was assayed using a specific chromogenic substrate. RESULTS: Our findings showed that the introduction and position of the hexahistidine tag did not affect the activity of the enzyme. However, the yield of the target protein was highest for the variant with seven C-terminal residues replaced by a hexahistidine sequence. CONCLUSION: We demonstrated the applicability of our approach for producing recombinant, enzymatically active 3Cpro.

Atypical glandular cells in Pap tests: cytology-histology correlation and risk assessment with human papillopmavirus (HPV) testing.

INTRODUCTION: Atypical glandular cells (AGC) represent less than 1% of Pap test cases and include a variety of lesions in both the cervix and endometrium. The study aimed to investigate the cytology-histology correlation in AGC patients and to evaluate the clinical utility of hrHPV testing in this diagnostic context. MATERIALS AND METHODS: We identified 491 atypical glandular cells (AGC) cases in our quality analysis (QA) database of 336,064 Pap tests interpreted between March 1, 2013 and July 12, 2016. Of these, 251 cases had follow-up biopsies with hrHPV tests in 148 cases. RESULTS: The most common histologic diagnosis associated with AGC was normal/benign or low-grade lesions, comprising 55% of cervical biopsies and 24% of endometrial biopsies. High-grade lesions were identified in 21% of follow-up biopsies. In patients with AGC cytology, a positive hrHPV test significantly increased the likelihood of cervical HSIL or above lesions on biopsy by 26.4 times (OR = 26.4, 95% CI: 5.8-119.4, P < 0.0001). A positive genotyping result for HPV 16 dramatically increased the likelihood of cervical HSIL or above lesions on biopsy (OR = 84, 95% CI: 12.0-590.5, P < 0.0001). The HPV test had a negative predictive value of 97% (CI: 85%-100%). CONCLUSIONS: Our study confirms that AGC is a significant diagnosis with an overall risk for high-grade cervical or endometrial lesions as high as 21%. hrHPV testing with genotyping is an effective tool for identifying high-risk individuals within the AGC population, with excellent positive and negative predictive values. This approach is valuable for clinical risk stratification and differential diagnosis in patients with AGC cytology.

Stability Study of a Compounded Sublingual Buprenorphine Solution for Neonatal Opioid Withdrawal Syndrome.

OBJECTIVE: Sublingual (SL) buprenorphine is a cornerstone of care in the treatment of adult opioid use disorder. Recent studies have demonstrated its advantages in the management of neonatal opioid withdrawal syndrome (NOWS). Commercially available SL tablets and transdermal patches are not amenable to neonatal use, and published compounding formulas of SL solutions contained undesirable excipients, including ethanol, sugars, and preservatives. The objective of this research is to explore the stability of a novel SL buprenorphine formulation free of alcohol, sugars, and preservatives. METHODS: A 0.075 mg/mL buprenorphine solution was prepared by diluting the commercial injectable solution with normal saline and packaged into polyethylene terephthalate amber prescription bottles and polypropylene amber oral syringes and stored in refrigeration. Quality assessments were conducted by visual, pH, and high-performance liquid chromatography (HPLC) analysis immediately after preparation, and at 7 and 14 days of storage. RESULTS: There were neither visual nor pH changes detected through 14 days. HPLC analysis indicated that all samples retained >99% initial buprenorphine concentration. Drug concentration increased slightly in the oral syringe after day 7, probably due to moisture loss. No degradation peaks were observed in chromatograms. CONCLUSIONS: This novel buprenorphine is free of alcohol, sugar, and preservatives, and it may offer a significant safety advantage for NOWS patients. Additional clinical studies are recommended to verify the bioavailability and efficacy of this formulation.

Lutein Modulates Oxidative Stress, Inflammatory and Apoptotic Biomarkers Related to Di-(2-Ethylhexyl) Phthalate (DEHP) Hepato-Nephrotoxicity in Male Rats: Role of Nuclear Factor Kappa B.

Phthalates are widely distributed in our environment due to their usage in many industries, especially in plastic production, which has become an essential part of daily life. This investigation aimed to assess the potential remedial influence of lutein, a naturally occurring carotenoid, on phthalate-triggered damage to the liver and kidneys. When di-(2-ethylhexyl) phthalate (DEHP) was administered to male albino rats over sixty straight days at a dosage of 200 mg/kg body weight, it resulted in a significant increase in the serum activity of liver enzymes (AST, ALT, and GGT), alpha-fetoprotein, creatinine, and cystatin-C, as well as disruptions in the serum protein profile. In addition, intoxication with DEHP affected hepato-renal tissues' redox balance. It increased the content of some proinflammatory cytokines, nuclear factor kappa B (Nf-κB), and apoptotic marker (caspase-3); likewise, DEHP-induced toxicity and decreased the level of anti-apoptotic protein (Bcl-2) in these tissues. Lutein administration at a dose level of 40 mg/kg b.w efficiently facilitated the changes in serum biochemical constituents, hepato-renal oxidative disturbance, and inflammatory, apoptotic, and histopathological alterations induced by DEHP intoxication. In conclusion, it can be presumed that lutein is protective as a natural carotenoid against DEHP toxicity.

Cerebrolysin as an Early Add-on to Reperfusion Therapy: Risk of Hemorrhagic Transformation after Ischemic Stroke (CEREHETIS), a prospective, randomized, multicenter pilot study.

BACKGROUND: Cerebrolysin could mitigate reperfusion injury and hemorrhagic transformation (HT) in animal models of acute ischemic stroke. METHODS: This was a prospective, randomized, open-label, parallel-group with active control, multicenter pilot study. Cerebrolysin (30 mL/day over 14 days) was administered concurrently with alteplase (0.9 mg/kg) in 126 patients, whereas 215 control patients received alteplase alone. The primary outcomes were the rate of any and symptomatic HT assessed from day 0 to 14. The secondary endpoints were drug safety and functional outcome measured with the National Institutes of Health Stroke Scale (NIHSS) on day 1 and 14, and the modified Rankin scale (mRS) on day 90. Advanced brain imaging analysis was applied on day 1 and 14 as a marker for in vivo pharmacology of Cerebrolysin. RESULTS: Cerebrolysin treatment resulted in a substantial decrease of the symptomatic HT rate with an odds ratio (OR) of 0.248 (95% CI: 0.072-0.851; p = 0.019). No serious adverse events attributed to Cerebrolysin occurred. On day 14, the Cerebrolysin arm showed a significant decrease in the NIHSS score (p = 0.045). However, no difference in the mRS score was observed on day 90. A substantial improvement in the advanced brain imaging parameters of the infarcted area was evident in the Cerebrolysin group on day 14. CONCLUSIONS: Early add-on of Cerebrolysin to reperfusion therapy was safe and significantly decreased the rate of symptomatic HT as well as early neurological deficit. No effect on day 90 functional outcome was detected. Improvements in the imaging metrics support the neuroprotective and blood-brain barrier stabilizing activity of Cerebrolysin. TRIAL REGISTRATION: Name of Registry: ISRCTN. TRIAL REGISTRATION NUMBER: ISRCTN87656744 . Trial Registration Date: 16/02/2021.

Efficiency of conventional and nanoparticle oxytetracycline in treatment of clinical endometritis in postpartum dairy cows.

The incidence of clinical endometritis in dairy cows postpartum is one of the important reasons for financial losses in the dairy industry. The costs of treatment, milk losses, infertility, repeated breeding, and high annual culling rate of dairy cows present immediate losses in case of treatment failure. The commonly used therapeutic methods for clinical endometritis have not been successful nor have given definitive solutions to overcome the complications of the disease in dairy cows. Therefore, it was necessary to propose an innovative treatment program to overcome the reasons for the failure and lack of effectiveness of the treatment of clinical endometritis. This was tackled in the current study; oxytetracycline with different concentrations, oxytetracycline 5% (OTCC5%), oxytetracycline 20% (OTCC20%), and oxytetracycline 20% nanoparticles (OTC-NPs) were used for the treatment of clinical endometritis. Diagnosis of clinical endometritis was based on the assessment of high serum concentration of pro-inflammatory cytokines, acute phase protein, increased endometrium thickness, and intrauterine discharges with different degrees of echogenicity monitored by ultrasonography. Application of OTC-NPs revealed a decrease in serum concentration of pro-inflammatory cytokines (IL-1, IL-6, and TNF-α) and acute phase proteins compared to OTCC20% and OTCC5% groups. The improvement achieved by OTC-NPs may be attributed to the reduction of OTC particles into nano size which facilitates its tissue bioavailability, dispersion, penetration power to deeper tissues, and its more broad-spectrum activities. These activities were clearly apparent after the evacuation of uterine contents using a single dose of PGF2α. The OTC-NPs revealed a reduction in serum concentration of cytokines compared to OTCC20% and OTCC5% groups arranged as follows: 10.11, 25.45, 35.56 for IL-1; 99, 300, 319 for IL-6; 1.01, 4.40, 8.06 for CRP; and 46, 183, 266 for TNF-α. Furthermore, an increase in serum concentration of albumin (3.34) was obtained by OTC-NPs compared to OTCC5% (1.70). This improvement can be taken as evidence of liver resumption functions and inflammatory reactions. On the other side, globulin concentration recorded an increase like albumin and total proteins in OTC-NPs compared to others. A reduction in the endometrium thickness in OTC-NPs with the disappearance of intrauterine discharges was monitored by ultrasonography. This confirmed the subsiding of clinical endometritis in OTC-NPs group. Moreover, a significant improvement in conception and pregnancy rate in OTC-NPs compared to other groups were observed.

Comparative study between two different morphological structures based on polylactic acid, nanocellulose and magnetite for co-delivery of flurouracil and curcumin.

Cellulose was extracted from mango fibers and subjected to acid hydrolysis to obtain a nanofiber. Two morphological structures based on the polylactic acid (PLA)/nanocellulose (NC) combination have been synthesized and Fe3O4 NPs (M) are incorporated into both combinations. The first formulation is obtained by blending technique (PLA/M-NC) and the second formulation is obtained by self-assembly of grafted copolymer (M-PLA-co-NC). The magnetic nanocomposites are used as carriers for 5-fluorouracil (5-FU), an anti-cancer drug, and curcumin (CUR) to get PLA/M-NC/5-FU/CUR and M-PLA-co-NC/5-FU/CUR. The structural, morphological, and magnetic properties of the obtained nanocomposites were characterized by various techniques. The loading, release of 5-FU/CUR and the inhibition efficacy of nanocarriers loaded drugs against bacteria, HePG-2, MCF-7, and HCT-116 cell lines were studied. The two morphological forms of nanocarriers are considered close in loading % of 5-FU; however, the M-PLA-co-NC nanocarrier loaded double the loading % of CUR into PLA/M-NC nanocarrier, revealing superiority of copolymeric micelle than the blended formulation. The dual drugs loaded magnetic copolymeric micelles M-PLA-co-NC/5-FU/CUR revealed slower release, higher antibacterial and antitumor efficacy than the PLA/M-NC/5-FU/CUR. In this respect, the M-PLA-co-NC/5-FU/CUR could be considered a good nanomedicine against Streptococcus, Bacillus subtilis, Klebsiella pneumonia and Escherichia coli bacteria, besides the investigated cell lines.

Screening Infants Through Adolescents for Social/Emotional/Behavioral Problems in a Pediatric Network.

OBJECTIVE: To assess changes in screening completion in a diverse, 7-clinic network after making annual screening for social/emotional/behavioral (SEB) problems the standard of care for all infant through late adolescent-aged patients and rolling out a fully automated screening system tied to the electronic medical record and patient portal. METHODS: In 2017, the Massachusetts General Hospital made SEB screening using the age-appropriate version of the Pediatric Symptom Checklist the standard of care in its pediatric clinics for all patients aged 2.0 months to 17.9 years. Billing records identified all well-child visits between January 1, 2016 and December 31, 2019. For each visit, claims were searched for billing for an SEB screen and the electronic data warehouse was queried for an electronically administered screen. A random sample of charts was reviewed for other evidence of screening. Chi-square analyses and generalized estimating equations assessed differences in screening over time and across demographic groups. RESULTS: Screening completion (billing and/or electronic) significantly increased from 2016 (37.2%) through 2019 (2017 [46.2%] vs 2018 [66.8%] vs 2019 [70.9%]; χ2 (3) =112652.33, P < .001), with an even higher prevalence found after chart reviews. Most clinics achieved screening levels above 90% by the end of 2019. Differences among demographic groups were small and dependent on whether data were aggregated at the clinic or system level. CONCLUSIONS: Following adoption of a best-practice policy and implementation of an electronic system, SEB screening increased in all age groups and clinics. Findings demonstrate that the AAP recommendation for routine psychosocial assessment is feasible and sustainable.

Evaluation of CDCP1 (CD318) and endoglin (CD105) expression as prognostic markers in acute myeloid leukemia.

BACKGROUND: The most commonly used prognostic factors in acute myeloid leukemia (AML) are cytogenetic, molecular, and morphological markers. However, AML prognosis is still unfavorable particularly in adults. So, further reliable markers are urgently needed to improve the risk stratification and treatment decisions. CUB domain-containing protein 1 (CDCP1; CD318) and endoglin (CD105) are new markers correlated with poor prognosis in different solid tumors, but their role in AML prognosis is not fully evaluated. OBJECTIVES: This work aimed to evaluate the prognostic role of CD318 and CD105 in AML and their impact on the outcomes. METHODS: Sixty-five newly diagnosed AML patients were included in this study. CD318 and CD105 expression was assessed by quantitative real-time polymerase chain reaction. Patients were followed up for ∼ 2 years to evaluate the prognostic impact of gene expression on the outcomes. RESULTS: Patients with high CD318 and CD105 showed higher white blood cell (WBC) count, M2 subtype, poor cytogenetic risk, reduced complete remission, and a greater number of deaths compared to low CD318 and CD105. CD318 was correlated with CD105, and both were correlated with WBC count, bone marrow blasts, and peripheral blood blasts. After a follow-up period of up to 24 months, relapse-free survival for high CD318 and CD105 was significantly different (42.1% and 52.6% vs. 64.5% and 58.1% for low CD318 and CD105, respectively). Survival was worse in patients with high CD318 and CD105, as the mean survival time was 13.9 and 13.3 months compared to 24 and 22.7 months in low CD318 and CD105, respectively. CONCLUSIONS: CD318 and CD105 are upregulated in AML patients. Their overexpression was associated with poor response to treatment and poor outcomes. Therefore, CD318 and CD105 can be useful prognostic markers in AML.

Rescreening of high-risk HPV positive Papanicolaou tests initially screened as negative is a low yield procedure in the era of HPV genotyping.

INTRODUCTION: Many laboratories rescreen Papanicolaou test slides initially interpreted as negative, but positive for human papillomavirus (HPV) high-risk types, as a quality control measure. We have evaluated the utility of this practice in the era of HPV genotyping as a laboratory improvement project. MATERIAL AND METHODS: Between August 2016 and October 2019, we identified 3618 rescreened Papanicolaou tests with follow-up biopsies. The biopsy results were put into 3 groups: 1) Negative; 2) LSIL: HPV changes or low-grade squamous intraepithelial lesion; and 3) HSIL: high-grade squamous intraepithelial lesion or carcinoma. HPV molecular testing results with subtyping for types 16 and 18 were available for 3117 of these cases. RESULTS: A total of 530 of 2812 Papanicolaou tests (18.8%) with positive HPV results were reinterpreted as cytologically abnormal after rescreening; 75 (14.2%) had a biopsy result of HSIL. The subset positive for HPV types 16/18 had 38 of 133 cytology positive cases diagnosed as HSIL on biopsy vs. 107 of 935 cytology negative cases diagnosed as HSIL on biopsy (28.6% vs. 11.4%, P < 0.0001). The subset positive for "other" (non-16/18) high-risk HPV types had 37 of 397 cytology positive follow-up HSIL vs. 84 of 1288 cytology negative follow-up HSIL (9.3% vs. 6.5%, P = 0.075). CONCLUSIONS: Rescreening has the highest yield in specimens positive for types 16/18. However, for this group colposcopy is recommended regardless of cytology findings, reducing the patient benefit. Routine rescreening of cytology negative/HPV positive Papanicolaou tests has reduced utility when HPV subtyping is performed and should be reconsidered.

Characterization of gut contractility and microbiota in patients with severe chronic constipation.

Chronic constipation (CC) is one of the most common gastrointestinal disorders worldwide. Its pathogenesis, however, remains largely unclear. The purpose of the present work was to gain an insight into the role of contractility and microbiota in the etiology of CC. To this end, we studied spontaneous and evoked contractile activity of descending colon segments from patients that have undergone surgery for refractory forms of CC. The juxta-mucosal microbiota of these colon samples were characterized with culture-based and 16S rRNA sequencing techniques. In patients with CC the spontaneous colonic motility remained unchanged compared to the control group without dysfunction of intestinal motility. Moreover, contractions induced by potassium chloride and carbachol were increased in both circular and longitudinal colonic muscle strips, thus indicating preservation of contractile apparatus and increased sensitivity to cholinergic nerve stimulation in the constipated intestine. In the test group, the gut microbiota composition was assessed as being typically human, with four dominant bacterial phyla, namely Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria, as well as usual representation of the most prevalent gut bacterial genera. Yet, significant inter-individual differences were revealed. The phylogenetic diversity of gut microbiota was not affected by age, sex, or colonic anatomy (dolichocolon, megacolon). The abundance of butyrate-producing genera Roseburia, Coprococcus, and Faecalibacterium was low, whereas conventional probiotic genera Lactobacillus and Bifidobacteria were not decreased in the gut microbiomes of the constipated patients. As evidenced by our study, specific microbial biomarkers for constipation state are absent. The results point to a probable role played by the overall gut microbiota at the functional level. To our knowledge, this is the first comprehensive characterization of CC pathogenesis, finding lack of disruption of motor activity of colonic smooth muscle cells and insufficiency of particular members of gut microbiota usually implicated in CC.

Functional efficiency of PCR vectors in vitro and at the organism level.

The functional efficiency of the expression cassettes integrated into a plasmid and a PCR- amplified fragment was comparatively analyzed after transient transfection in vitro or introduction into the developing embryo of Danio rerio. The cassettes contained the reporter genes, luciferase of Photinus pyralis (luc) or enhanced green fluorescent protein, under the control of the promoter of human cytomegalovirus immediate-early genes. In the in vitro system, the efficiency of the circular plasmid was 2.5 times higher than that of the PCR- amplified fragment. The effect of mutations in the expression cassette on the efficiency of the transgene expression in the PCR- amplified fragment was quantitatively evaluated. The mutations generated after 25 amplification cycles with Taq DNA polymerase decreased luciferase activity in transfected cells by 65-85%. Thus, mutations are the key factor of decreased functional efficiency of the PCR- amplified fragment relative to the circular plasmid in this experimental model, while other factors apparently have a lesser impact. At the organism level, no significant difference in the expression efficiency of the plasmid and PCR- amplified fragment has been revealed. Comparison of the vector efficiencies in in vivo and in vitro systems demonstrates that the level of luciferase in the D. rerio cell lysate, normalized to the molar concentration of the vector, is by three orders of magnitude higher than that after the cell transfection in vitro, which indicates that the quantitative data obtained for in vitro systems should not be directly extrapolated to the organism level.

Age-specific 3-year cumulative risk of cervical cancer and high-grade dysplasia on biopsy in 9434 women who underwent HPV cytology cotesting.

BACKGROUND: High-risk human papillomavirus (HPV)-Papanicolaou (Pap) cotesting is recommended for cervical cancer screening in women aged ≥30 years. The current study analyzed the effectiveness of cotesting on risk management in different age groups. METHODS: A retrospective review of a 5-year cytology database identified 9434 women with HPV-Pap cotesting and follow-up cervical biopsy. The 3-year cumulative risk of developing high-grade cervical lesions (≥high-grade squamous intraepithelial lesion [HSIL]) was analyzed using age stratification. RESULTS: The 3-year cumulative risk of developing ≥HSIL was found to be significantly different in women with baseline cotesting HPV-positive and Pap-positive results (HPV+/Pap+; defined as ≥atypical squamous cells of undetermined significance), HPV+ and Pap-negative results, and HPV-negative and Pap+ results at 19.2%, 7.9%, and 3.1%, respectively (P < .001). The risk of ≥HSIL peaked at ages 30 to 39 years and significantly decreased at ages 50 to 59 years (16.6% vs 6.7%; P < .001). Women aged <30 years shared a high risk similar to that of women aged 30 to 39 years (17.3% vs 16.6%; P = .52), and risk stratification by cotesting was found to be equally effective in the younger age group (HPV+ and Pap+: 19.6%; HPV+ and Pap-negative: 7.2%; and HPV-negative and Pap+: 4.4% [P < .001]). CONCLUSIONS: High-risk HPV-Pap cotesting appears to be extremely sensitive for the prediction of the risk of developing ≥HSIL and is an effective tool for risk stratification. In the current study, the 3-year cumulative risk of developing ≥HSIL varied significantly with age, with the highest risk noted among women aged <40 years and the lowest risk observed in women aged 50 to 59 years. Pap testing significantly impacted risk stratification in the HPV+ positive group, especially in women aged <60 years. Women aged <30 years were found to have a risk profile and cotesting efficacy similar to those of women aged 30 to 39 years. Modification of the current recommendation to offer cotesting to women aged ≥30 years might be considered to include those patients aged <30 years.

PDX1, a key factor in pancreatic embryogenesis, can exhibit antimetastatic activity in pancreatic ductal adenocarcinoma.

Background: In cancer biology, metastasizing is one of the most poorly studied processes. Pancreatic ductal adenocarcinoma (PDAC) is characterized by early metastasis, which is the leading cause of death. The PDX1 protein is crucial for the development of cancer, and its low levels are characteristic of the most aggressive PDAC tumors. The PDX1 is a mediator of initiation and progression of PDAC. However, further studies are needed to elucidate the role of PDX1 in the cancer metastasis. Purpose: To confirm the hypothesis that PDX1 in PDAC plays suppressor role of epithelial-mesenchymal transition (EMT), and to study its possible ability to inhibit metastasis. Methods: A PDX1-overexpressing PDAC cell line was obtained by lentiviral transduction of PANC-1 cells. PDX1 overexpression was confirmed by RT-PCR and Western blotting. Effects of PDX1 ectopic expression on cell proliferation and motility were determined in PANC-1 cells using MTS, cell cycle analysis, transwell and wound-healing assay. EMT genes expression was analyzed in PDX1-overexpressing and Control PANC-1. Finally, the migration potential of pancreatic cancer cells expressing PDX1 was evaluated using a zebrafish embryo model. Results: The motility of human PDAC cells PANC-1 considerably decreased at ectopic expression of PDX1. The decreased expression of ZEB1, the key factor of EMT, and almost unchanged expression of the genes that characterize the epithelial state suggest a decrease in the EMT ability. Suppression of PDX1 expression by siRNA knockdown restored the PANC1 motility. Conclusion: The results obtained suggest a possible therapeutic use of PDX1 delivery into PDAC patients with a reduced or absent expression of PDX1 in the most aggressive tumors.

Attenuation potentials of royal jelly against hydroxyurea-induced infertility through inhibiting oxidation and release of pro-inflammatory cytokines in male rats.

Hydroxyurea (HDU), a class of antineoplastic drugs, has a powerful efficacy in the treatment of several types of malignancies. However, it has multiple adverse effects including reduced fertility, especially in males. Thus, 60 male albino rats were used to investigate the chemoprotective potentials of royal jelly on HDU-induced testicular damage. Animals were gastro-gavaged with HDU (225 or 450 mg kg-1 bw day-1) before royal jelly (100 mg kg-1 bw day-1) for 60 days. Blood samples and testicles were collected, and spermatozoon was obtained. In a dose-dependent manner, the sperm count, motility and liveability, and testosterone, GSH, and catalase concentrations were decreased in HDU groups, whereas MDA, FSH, LH, IL-6, and IFN-γ expression levels were increased. Germinal epithelium degeneration, germ cell sloughing, reduction in the number of luminal spermatozoa, interstitial congestion, and severe leukocyte infiltration besides no glandular secretion in most of the acini were identified. However, royal jelly intake in HDU-treated rats successfully improved sperm quality, hormonal and antioxidant status, and reproductive organ histoarchitecture. Thus, it could be concluded that royal jelly is endowed with antioxidative and anti-inflammatory activities and could be, therefore, used as an adjuvant remedy to improve HDU-induced male subfertility.

HPV status in women with high-grade dysplasia on cervical biopsy and preceding negative HPV tests.

INTRODUCTION: A considerable number of patients with high-grade cervical lesions have undergone preceding human papillomavirus (HPV) tests with negative results. In the present study, we attempted to elucidate the factors potentially contributing to the findings by testing biopsied samples from these patients. MATERIALS AND METHODS: Of the 1654 women with HPV testing and follow-up cervicovaginal biopsies from March 1, 2013 to June 30, 2014, 21 of 252 women (8.3%) with biopsy-confirmed high-grade squamous intraepithelial lesion (HSIL) or worse had had negative results from preceding high-risk (hr)HPV tests. The corresponding paraffin blocks were tested for HPV using the Cobas 4800 system, a DNA microarray against 40 HPV genotypes, and DNA sequencing. RESULTS: HPV was detected in 20 (95%) of the 21 biopsies with HSIL or worse, including HPV16/18 in 4, non-16/18 hrHPV in 10, and non-hrHPV in 6. HPV59 and HPV45 were 2.2 times more frequently detected than HPV16/18 in these samples. One sample was negative for all 3 tests (5%). CONCLUSIONS: Our study has demonstrated that 8.3% of women with biopsy-confirmed HSIL or worse had preceding test results that were negative for hrHPV. The vast majority of the biopsied samples had detectable HPV, primarily hrHPV genotypes (67%) with HPV59 and HPV45 predominance. This genotypic prevalence pattern was markedly different from those reported in the general population. Non-hrHPV genotypes contributed to 29% of the cases, and HPV-negative cases were rare. In addition to the limited Cobas testing panel and rare possible HPV-negative HSIL or worse, other possible contributing factors to the discrepancy include cytologic sampling, interference material, technical errors, and reduced L1 gene expression in high-grade lesions.

Bethesda 2014 Implementation and Human Papillomavirus Primary Screening: Practices of Laboratories Participating in the College of American Pathologists PAP Education Program.

CONTEXT.­: Cervical cancer screening laboratory practices may evolve with new terminology and technologies. OBJECTIVE.­: To investigate changes in cervical cytopathology practice resulting from the 2014 Bethesda System updates and screening technologies. DESIGN.­: Questionnaires accompanied 2016 and 2017 mailings of the College of American Pathologists PAP Education program. RESULTS.­: In 2016, most laboratories surveyed had adopted or were planning to adopt 2014 Bethesda System updates, and the majority (53%; 365 of 689) used an age cutoff of 45 for reporting benign-appearing endometrial cells. However, 51.3% (354 of 690) of laboratories used the term low-grade squamous intraepithelial lesion, cannot exclude high-grade squamous intraepithelial lesion, for cases with indeterminate features, and 44.9% (298 of 664) of laboratories used a 5000-cell cutoff for minimum squamous cellularity for posthysterectomy and posttherapy specimens. Reporting rates for cervical cytology metrics changed very little from 2013 to 2016, and the median ratio of atypical squamous cells to squamous intraepithelial lesion cases was 1.9 for ThinPrep and 1.8 for SurePath preparations. Most laboratories (59.4%; 389 of 655) did not offer stand-alone primary human papillomavirus (HPV) testing in 2017, and primary HPV testing accounted for a low proportion of HPV testing volumes. The Roche Cobas method was the most common platform for HPV primary screening. CONCLUSIONS.­: These questionnaire surveys provide data about the current status of cervical cytology screening, including changes related to the 2014 Bethesda System updates and the adoption of HPV primary screening techniques.

Intrinsic Functional Brain Connectivity Predicts Onset of Major Depression Disorder in Adolescence: A Pilot Study.

Children with familial risk for major depressive disorder (MDD) have elevated risk for developing depression as adolescents. Here, we investigated longitudinally whether resting-state functional connectivity (RSFC) could predict the onset of MDD. In this pilot study, we followed a group of never-depressed children with familial risk for MDD and a group of age-matched controls without familial risk who had undergone an MRI study at 8-14 years of age. Participants were reassessed 3-4 years later with diagnostic interviews. We first investigated group differences in RSFC from regions in the emotion regulation, cognitive control, and default mode networks in the children who later developed MDD (converted), the children who did not develop MDD (nonconverted), and the control group. We then built a prediction model based on baseline RSFC that was independent of the group differences to classify the individuals who later developed MDD. Compared with the nonconverted group, the converted group exhibited hypoconnectivity between subgenual anterior cingulate cortex (sgACC) and inferior parietal lobule (IPL) and between left and right dorsolateral prefrontal cortices. The nonconverted group exhibited higher sgACC-IPL connectivity than did both the converted and control groups, suggesting a possible resilience factor to MDD. Classification between converted and nonconverted individuals based on baseline RSFC yielded high predictive accuracy with high sensitivity and specificity that was superior to classification based on baseline clinical rating scales. Intrinsic brain connectivity measured in healthy children with familial risk for depression has the potential to predict MDD onset, and it can be a useful neuromarker in early identification of children for preventive treatment.

Role of HPV genotyping in risk assessment among cytology diagnosis categories: analysis of 4562 cases with cytology-HPV cotesting and follow-up biopsies.

OBJECTIVE: Human papilloma virus (HPV) detection and genotyping are increasingly used in clinical risk assessment. We aimed to analyze HPV genotyping performance in risk stratification among cytology diagnosis categories. METHODS: Between January 1, 2015 and December 31, 2016, 4562 cases with cytology-HPV co-testing and biopsy follow-up were identified. HPV tests were performed on Cobas (n=3959) or Aptima (n=603) platforms. Of the biopsies, 669 demonstrated high-grade squamous intraepithelial lesions or worse. RESULTS: Pooled high-risk HPV testing had high overall sensitivity (97%) but low specificity (20%) and positive predictive value (20%) for biopsy-confirmed high-grade squamous intraepithelial lesions or worse. HPV16/18 genotyping had considerably improved specificity (81%) and positive predictve value (35%) in predicting high-grade squamous intraepithelial lesions or worse, especially in atypical squamous cells of undetermined significance and low-grade squamous intraepithelial lesion categories. Significantly more biopsy-confirmed high-grade squamous intraepithelial lesions or worse were detected by Aptima than Cobas testing, as measured by HPV16/18 (48% vs 33%, p<0.001), non-16/18 high-risk HPV (18% vs 13%, p=0.029), or all high-risk HPV genotypes (27% vs 19%, p<0.001). Aptima genotyping showed a significantly higher positive predictive value than Cobas genotyping for biopsy-confirmed high-grade squamous intraepithelial lesions or worse in the atypical squamous cells of undetermined significance category (47% vs 23%, p<0.05). CONCLUSIONS: HPV genotyping was sensitive for biopsy-confirmed high-grade squamous intraepithelial lesions or worse in all cytologic categories, and is particularly valuable in risk evaluation for women with atypical squamous cells of undetermined significance and low-grade squamous intraepithelial lesions. The triaging role was greatly diminished in high-risk lesions (atypical glandular cells, atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesions and high-grade squamous intraepithelial lesions) due to low specificity and positive predictive value. Aptima performance in risk management was superior to Cobas, with significantly higher positive predictive value for biopsy-confirmed high-grade squamous intraepithelial lesions or worse. Our results highlight the importance of careful data interpretation from studies using different HPV testing methods and the need to incorporate HPV E6/E7-mRNA testing into management guidelines.

Early screening for gestational diabetes mellitus is not associated with improved pregnancy outcomes: an observational study including 9795 women.

AIMS: In addition to screening for hyperglycaemia during pregnancy after 24 weeks of gestation (WG), the current guidelines also suggest screening in early pregnancy and referring women with early gestational diabetes mellitus (eGDM) or overt diabetes (OD) for immediate care. Our aim was to evaluate this strategy. METHODS: This study evaluated, at our hospital (2012-2016), whether the incidence of a predefined composite outcome (preeclampsia, large-for-gestational-age infant, shoulder dystocia) and secondary outcomes was different when women were screened only after 22WG ('late screening only') or before 22WG and treated for eGDM or OD if present, with repeat screening after 22WG if absent ('early ± late screening'). RESULTS: Early ± late screening (n = 4605, 47.0%) increased between 2012 and 2016 (P < 0.0001) and was associated with more risk factors for GDM than late screening only. Glycaemic status differed in both groups (early ± late screening: eGDM 10.3%, GDM 12.1%, OD 0.9% vs. late screening only: GDM 16.8%, OD 1.2%; P < 0.001), with a higher rate of insulin therapy (8.9% vs. 6.0%; P < 0.001) and less gestational weight gain (11.1 ± 5.4 kg vs. 11.4 ± 5.5 kg; P = 0.013) in the early ± late screening group. Rates of those meeting the composite criterion were similar in both groups [11.6% vs. 12.0%, respectively; odds ratio (OR): 1.040, 95% confidence interval (CI): 0.920-1.176; P = 0.53] and remained comparable after adjusting for Propensity Scores (OR: 1.046, 95% CI: 0.924-1.185; P = 0.4790). Rates for secondary outcomes were also similar in both groups. CONCLUSION: While a strategy including early measurement of fasting plasma glucose during pregnancy increases the incidence and care of hyperglycaemia during pregnancy, it may not significantly improve pregnancy outcomes.