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ABSTRACTMachine learning methods have been extensively employed to predict COVID-19 using chest X-ray images in numerous studies. However, a machine learning model must exhibit robustness and provide reliable predictions for diverse populations, beyond those used in its training data, to be truly valuable. Unfortunately, the assessment of model generalisability is frequently overlooked in current literature. In this study, we investigate the generalisability of three classification models – ResNet50v2, MobileNetv2, and Swin Transformer – for predicting COVID-19 using chest X-ray images. We adopt three concurrent approaches for evaluation: the internal-and-external validation procedure, lung region cropping, and image enhancement. The results show that the combined approaches allow deep models to achieve similar internal and external generalisation capability.

Background:There is scarce data on the level of handicap in Parkinson’s disease (PD) and none in advanced stage PD. Objective:To assess the handicap in advanced stage PD patients with disabling levodopa-induced motor complications selected to deep brain stimulation (DBS). Methods:Data was prospectively recorded during routine evaluation for DBS. Handicap was measured using London Handicap Scale (LHS) (0 = maximal handicap; 1 = no handicap). Disease severity was evaluated using the Hoehn & Yahr scale and the UPDRS/MDS-UPDRS, during offand onafter a supra-maximal dose of levodopa. Schwab and England Scale (S&E) was scored in offand on. Dyskinesias were scored using the modified Abnormal Involuntary Movement Scale (mAIMS). Results concern cross-sectional assessment before DBS. Results:100 PD patients (mean age 61 (±7.6); mean disease duration 12.20 (±4.6) years) were included. Median score of motor MDS-UPDRS was 54 in offand 25 in on.Mean total LHS score was 0.56 (±0.14). Patients were handicapped in several domains with a wide range of severity. Physical Independence and Social Integration were the most affected domains. Determinants of total LHS score were MDS-UPDRS part II off(β= –0.271; p = 0.020), S&E on(β= 0.264; p = 0.005) and off(β= 0.226; p = 0.020), and mAIMS on(β= –0.183; p = 0.042) scores (R2 = 29.6%). Conclusions:We were able to use handicap to measure overall health condition in advanced stage PD. Patients were moderately to highly handicapped and this was strongly determined by disability in ADL and dyskinesias. Change in handicap may be a good patient-centred outcome to assess efficiency of DBS.

The intestine is extremely sensitive to ischemic preservation and reoxygenation injury. Current vascular perfusion and cold storage with University of Wisconsin (UW) solution neglect the intestinal lumen and the ongoing mucosal metabolism during hypothermia. This study was designed to test the effects of luminal preservation with an alternative preservation solution in addition to the common vascular flush with UW solution on graft viability after preservation and ex vivoreoxygenation.

Development of improved protocols for cryopreservation of zona pellucida-intact porcine embryos could greatly impact the swine industry. Our aim was to investigate in vitro development following cryopreservation of embryos from Chinese Meishan (M) and occidental white cross (WC) breeds using a modified protocol described previously (Misumi K et al. 2003 Theriogenology 60, 253?260). First-parity M sows (n = 11) and WC gilts (n = 13) were observed for estrus every 12 h and inseminated at 12 and 24 h after estrous onset within breed using semen from 2 different boars. Females were sacrificed between Days 4.5 and 6 after estrus and embryos were collected using Beltsville embryo culture medium (BECM). Compact morula (CM) or blastocyst stage embryos from each female within breed were randomly allocated either directly into the culture system to serve as controls (68 M and 48 WC embryos) or to undergo cryopreservation. A total of 101 M and 78 WC embryos were cryopreserved using the following protocol: (1) 5 min in BECM + 10% ethylene glycol (EG); (2) 5 min in BECM + 10% EG + 0.27 M sucrose + 1% polyethylene glycol (PEG); and (3) 30 to 45 s in BECM + 40% EG + 0.36 M sucrose + 2% PEG. In the last solution, 5 to 10 embryos in a 5- to 10-?L microdrop attached to a fine glass pipette were exposed to the vapor phase of liquid nitrogen (LN2) for 15 s and then plunged into LN2. The pipette tip was broken and the tip and associated frozen microdrop were placed inside an LN2-submerged 2-mL cryotube containing a hole in the lid for 1 h. Next, embryos were thawed using a 4-step (5 min each) procedure: (1) BECM + 5% EG + 0.57 M sucrose; (2) BECM + 2.5% EG + 0.29 M sucrose; (3) BECM + 0.3 M sucrose; and (4) BECM alone. All procedures were performed with solutions maintained at 37C. Cryopreserved and control embryos were cultured in 50 ?L drops of modified Whitten's medium + 1.5% BSA under oil at 37C in a 5% CO2 in air environment and scored daily for development. For embryos undergoing cryopreservation, retrieval rates from cryovials were 92% and 96% for M and WC, respectively. The percentage of embryos surviving 24 h after cryopreservation without lysis or degeneration was higher for M (72%) than for WC (44%; P < 0.001; ?2-test). However, in vitro development of embryos that survived cryopreservation was not different between M and WC at the expanded (64%) or hatched (22%) blastocyst stages. Developmental rates were significantly higher for control embryos than for frozen embryos from both breeds. Rates of expanded blastocyst formation did not differ between M and WC control embryos (98% and 95%, respectively), but more M embryos developed to the hatched blastocyststage (22% for M v. 9% for WC; P < 0.05). Our results suggest that M embryos have a higher capacity to survive the vitrification process than WC embryos.  Funding for M. Montagner was provided by CAPES, Brazil.

Chronic renal injury can be mediated by angiotensin II (ANG II) and prostanoids through hemodynamic and inflammatory mechanisms and attenuated by individual suppression of these mediators. In rats with (5/6) renal ablation (Nx), we investigated 1) the intrarenal distribution of COX-2, ANG II, and the AT(1) receptor (AT(1)R); 2) the renoprotective and antiinflammatory effects of an association between the AT(1)R blocker, losartan (Los), and the gastric sparing anti-inflammatory nitroflurbiprofen (NOF). Adult male Munich-Wistar rats underwent Nx or sham operation (S), remaining untreated for 30 days, after which renal structure was examined in 12 Nx rats (Nx(pre)). The remaining rats were followed during an additional 90 days, distributed among 4 treatment groups: Nx(V) (vehicle), Nx(Los) (Los), Nx(NOF) (NOF), and Nx(Los/NOF) (Los/NOF). Nx(pre) rats exhibited marked albuminuria, hypertension, glomerulosclerosis, interstitial expansion, and macrophage infiltration, accompanied by abnormal glomerular, vascular, and interstitial COX-2 expression. ANG II appeared in interstitial cells, in contrast to S, in which ANG II was virtually confined to afferent arterioles. Intrarenal AT(1)R distribution shifted from mostly tubular in S to predominantly interstitial in Nx(pre). All these changes were aggravated at 120 days and attenuated by Los and NOF monotherapies. Los/NOF treatment arrested renal structural injury and ANG II expression and reversed hypertension, albuminuria, and renal inflammation. In conclusion, abnormal expression of COX-2, ANG II, and AT(1)R may be key to development of renal injury in Nx. Concomitant COX-2 inhibition and AT(1)R blockade arrested renal injury and may represent a useful strategy in the treatment of chronic nephropathies.

Development of improved protocols for cryopreservation of zona pellucida-intact porcine embryos could greatly impact the swine industry. Our aim was to investigate in vitro development following cryopreservation of embryos from Chinese Meishan (M) and occidental white cross (WC) breeds using a modified protocol described previously (Misumi K et al. 2003 Theriogenology 60, 253?260). First-parity M sows (n = 11) and WC gilts (n = 13) were observed for estrus every 12 h and inseminated at 12 and 24 h after estrous onset within breed using semen from 2 different boars. Females were sacrificed between Days 4.5 and 6 after estrus and embryos were collected using Beltsville embryo culture medium (BECM). Compact morula (CM) or blastocyst stage embryos from each female within breed were randomly allocated either directly into the culture system to serve as controls (68 M and 48 WC embryos) or to undergo cryopreservation. A total of 101 M and 78 WC embryos were cryopreserved using the following protocol: (1) 5 min in BECM + 10% ethylene glycol (EG); (2) 5 min in BECM + 10% EG + 0.27 M sucrose + 1% polyethylene glycol (PEG); and (3) 30 to 45 s in BECM + 40% EG + 0.36 M sucrose + 2% PEG. In the last solution, 5 to 10 embryos in a 5- to 10-?L microdrop attached to a fine glass pipette were exposed to the vapor phase of liquid nitrogen (LN2) for 15 s and then plunged into LN2. The pipette tip was broken and the tip and associated frozen microdrop were placed inside an LN2-submerged 2-mL cryotube containing a hole in the lid for 1 h. Next, embryos were thawed using a 4-step (5 min each) procedure: (1) BECM + 5% EG + 0.57 M sucrose; (2) BECM + 2.5% EG + 0.29 M sucrose; (3) BECM + 0.3 M sucrose; and (4) BECM alone. All procedures were performed with solutions maintained at 37C. Cryopreserved and control embryos were cultured in 50 ?L drops of modified Whitten's medium + 1.5% BSA under oil at 37C in a 5% CO2 in air environment and scored daily for development. For embryos undergoing cryopreservation, retrieval rates from cryovials were 92% and 96% for M and WC, respectively. The percentage of embryos surviving 24 h after cryopreservation without lysis or degeneration was higher for M (72%) than for WC (44%; P < 0.001; ?2-test). However, in vitro development of embryos that survived cryopreservation was not different between M and WC at the expanded (64%) or hatched (22%) blastocyst stages. Developmental rates were significantly higher for control embryos than for frozen embryos from both breeds. Rates of expanded blastocyst formation did not differ between M and WC control embryos (98% and 95%, respectively), but more M embryos developed to the hatched blastocyststage (22% for M v. 9% for WC; P < 0.05). Our results suggest that M embryos have a higher capacity to survive the vitrification process than WC embryos.  Funding for M. Montagner was provided by CAPES, Brazil.

Osteopontin (OPN), a secretory RGD-containing phosphoprotein, has been identified in cow oviductal ephitelium and fluid, but its role in fertilization is unclear. RGD peptide is capable of blocking fertilization, inducing intracellular Ca2+ transients, and initiating parthenogenetic development when present during bovine fertilization in vitro.This study was conducted to determine whether in vitro sperm binding to the zona pellucida (ZP) and fertilization of bovine oocytes were affected by treating the sperm or oocytes with RGD (arginine-glycine-aspartic acid, a sequence recognized by integrins) or non-RGD-containing peptides. In vitro matured oocytes were incubated (39C, 5% CO2 in air) for 2 hours in fertilization medium with: (1) no peptides;; (2) 50gmL-1 RGD (Calbiochem, San Diego, CA, USA); (3) 1000gmL-1; (4) 50gmL-1 non-RGD (Calbiochem); (5) 1000gmL-1 non-RGD. The bovine sperm from two differents bulle was collected by artificial vagina, pooled, washed twice with MTM at 500g for 10min and incubated (39C, 5% CO2 in air) for two h at 5107 concentration in MTM with: (6) no peptides;; (7) 50gmL-1 RGD;; (8) 1000gmL-1; (9) 50gmL-1 non-RGD; (10) 1000gmL-1 non-RGD. Following incubation, treated and untreated oocytes were washed and inseminated with 1105 treated or untreated fresh spermatozoa per 10 oocytes;; after the sperm were recovered from a Percoll gradient (45%/90%). After 18-20h, oocytes were removed from co-culture, and washed in TL-HEPES. Oocytes used to assess sperm binding were stained with Hoescht 33342, and the number of sperm bound per ZP counted. The remaining oocytes were fixed in acid alcohol, stained with 1% acetato-orcein and observed for the presence of pronuclei. For the five replicates, 100-120 oocytes were used for fertilization and 150-170 oocytes were used for sperm-egg binding assays. Data were analized by SAS. Treatment of sperm or oocytes with the RGD peptide significantly decreased (P&lt;0.05) fertilization compared to the non-treated controls or those treated with non-RGD peptides: (1) 80%3.0; (2) 42%3.0; (3) 30.2%3.0; (4) 78.5%3.0; (5) 79.1%3.0; (6) 78.9%3.0; (7) 41.3%3.0; (8) 29.1%3.0; (9) 79.2%3.0; (10) 80.2%3.0. More sperm bound to the ZP of untreated or non-RGD-treated oocytes or sperm than those incubated with the RGD peptide: (1) 71.24.1; (2) 33.24.2; (3) 24.24.1; (4) 69.54.1; (5) 70.24.2; (6) 71.94.2; (7) 29.84.2; (8) 19.84.2; (9) 68.94.2; (10) 70.64.2. These studies demonstrated that incubation of bovine oocytes or spermatozoa with a RGD peptide inhibits sperm-egg binding and fertilization in vitro. These findings support the notion that the role of osteopontin in bovine fertilization may involve inteaction with integrins via its RGD sequence.