Development and Validation of Three Automated High-Throughput Molecular Tests to Detect Monkeypox Virus Infections.

BACKGROUND: The 2022 outbreak of the clade IIb monkeypox virus and subsequent global spread lead to an urgent need for the development of high-throughput, sensitive, and reproducible diagnostic tests. METHODS: We developed 3 assays to detect monkeypox virus, 2 (MPXV+ and MPXV) for m2000 RealTime and 1 (MPXV) for Alinity m platforms. Dual targets in E9L and B6R (MPXV+) and J2L and B7R (MPXV) increased mutation resistance. In silico prediction indicates MPXV+ cross-reactivity with orthopox viruses and specific monkeypox virus detection with MPXV. RESULTS: m2000 RealTime MPXV+ and MPXV assay sensitivity was determined to be 3.2 plaque-forming units/mL using a reference virus culture diluted into universal transport medium (UTM). Alinity m MPXV lower limit of detection was 200 copies/mL using monkeypox virus plasmids in pooled UTM matrix. m2000 RealTime MPXV+ and MPXV assays were validated with lesion swabs in UTM and 1:1 saliva to UTM mixtures. Commercially available and remnant clinical lesion specimens in UTM were tested with RealTime MPXV+, RealTime MPXV and Alinity m MPXV assays and demonstrated high agreement to known mpox (MPX)-positive specimens. CONCLUSIONS: RealTime MPXV+, RealTime MPXV, and Alinity MPXV are high throughput and sensitive assays used for the detection of monkeypox virus. These assays maybe useful during MPX outbreaks.

EphA2- and HDAC-Targeted Combination Therapy in Endometrial Cancer.

Endometrial cancer is the most frequent malignant tumor of the female reproductive tract but lacks effective therapy. EphA2, a receptor tyrosine kinase, is overexpressed by various cancers including endometrial cancer and is associated with poor clinical outcomes. In preclinical models, EphA2-targeted drugs had modest efficacy. To discover potential synergistic partners for EphA2-targeted drugs, we performed a high-throughput drug screen and identified panobinostat, a histone deacetylase inhibitor, as a candidate. We hypothesized that combination therapy with an EphA2 inhibitor and panobinostat leads to synergistic cell death. Indeed, we found that the combination enhanced DNA damage, increased apoptosis, and decreased clonogenic survival in Ishikawa and Hec1A endometrial cancer cells and significantly reduced tumor burden in mouse models of endometrial carcinoma. Upon RNA sequencing, the combination was associated with downregulation of cell survival pathways, including senescence, cyclins, and cell cycle regulators. The Axl-PI3K-Akt-mTOR pathway was also decreased by combination therapy. Together, our results highlight EphA2 and histone deacetylase as promising therapeutic targets for endometrial cancer.

Combined cholecystotomy, retrograde hydropulsion, and choledochal stenting to treat extrahepatic biliary tract obstruction in 3 cats.

Extrahepatic biliary tract obstruction (EHBO) is uncommonly encountered in cats. Surgical treatment aims to decompress the biliary tract and insure bile duct patency. In veterinary medicine, cholecystotomy is not widely used in practice. The objective was to describe the use of cholecystotomy, retrograde hydropulsion of choleliths, and choledochal stenting to remove choleliths from the extrahepatic biliary tract back in the gallbladder. Three adult domestic shorthair cats were presented with anorexia, lethargy, and vomiting. Serum biochemistry revealed hyperbilirubinemia and increased hepatic enzymes. Abdominal ultrasonography showed evidence of EHBO requiring surgical intervention. Choleliths were localized in the proximal and middle portions of the common bile duct (CBD) in the first case, in the distal portion of the CBD and within the major duodenal papilla in the second case, and in the middle and distal portions of the CBD in the third case. Cholecystotomy was followed by retrograde hydropulsion of the choleliths into the gallbladder, after which choledochal stenting was performed. Complications were defined as major when requiring additional medical or surgical treatment, or minor when not. Three major complications were reported. In 2 cases, severe anemia requiring blood transfusion occurred 24 h postoperatively; in 1 case, EHBO recurrence was encountered 41 d postoperatively. All cats were discharged within 4 d following surgery. Two cats were still alive at 12 and 14 mo after surgery, respectively. In the last case, owners refused revision surgery and the cat was euthanized. Key clinical message: Cholecystotomy combined with retrograde hydropulsion of choleliths permitted removal of choleliths and decompression of the biliary tract in 3 cats. Major complications included severe anemia and EHBO recurrence.

Cholécystotomie combinée, hydropulsion rétrograde et pose de stent cholédocien pour traiter l'obstruction des voies biliaires extra-hépatiques chez 3 chats. Les obstructions biliaires extra-hépatiques (OBEH) sont peu fréquentes chez le chat. Le traitement chirurgical vise à lever l'obstruction et s'assurer de la perméabilité des voies biliaires. En médecine vétérinaire, la cholécystotomie est une technique peu pratiquée. L'objectif de ce rapport de cas était de décrire l'utilisation de la cholécystotomie, de l'hydropulsion rétrograde des cholélithes et d'une prothèse endoluminale cholédoquale (PEC) pour repousser les cholélithes présents dans les voies biliaires extrahépatiques dans la vésicule biliaire (VB).Trois chats européens adultes ont été présentés pour anorexie, léthargie et vomissements. La biochimie sérique a révélé une hyperbilirubinémie et une augmentation des enzymes hépatiques. L'échographie abdominale a mis en évidence une OBEH nécessitant une intervention chirurgicale. Les cholélithes étaient situés dans la portion proximale et moyenne du canal cholédoque pour le premier cas; dans la portion distale et la papille duodénale majeure dans le second cas; dans la portion moyenne et distale pour le troisième cas. Une cholécystotomie a été suivie d'une rétro-hydropulsion des cholélithes dans la VB, puis une PEC a été placée. Les complications ont été définies comme majeures lorsqu'elles nécessitaient un traitement médical ou chirurgical supplémentaire, ou mineures lorsqu'elles n'en nécessitaient pas.Trois complications majeures ont été rapportées : chez 2 cas, une anémie sévère a été observée 24 h après l'intervention, nécessitant une transfusion sanguine; chez un cas, une récidive d'obstruction biliaire a eu lieu à 41 jours postopératoire. Tous les patients sont sortis de l'hôpital dans les 4 jours suivant l'opération. Deux cas étaient encore en vie 12 et 14 mois après l'intervention. Pour le dernier cas, la seconde chirurgie a été refusée par les propriétaires et le chat a été euthanasié.Message clinique clé :La cholécystotomie combinée à l'hydropulsion rétrograde des cholélithes a permis le retrait de cholélithes obstructives (dont certaines distales) et la décompression du tractus biliaire chez 3 chats. Les complications majeures incluaient une anémie sévère et une récidive d'obstruction biliaire.(Traduit par les auteurs).

Filtering input fluctuations in intensity and in time underlies stochastic transcriptional pulses without feedback.

Stochastic pulsatile dynamics have been observed in an increasing number of biological circuits with known mechanism involving feedback control and bistability. Surprisingly, recent single-cell experiments in Escherichia coli flagellar synthesis showed that flagellar genes are activated in stochastic pulses without the means of feedback. However, the mechanism for pulse generation in these feedbackless circuits has remained unclear. Here, by developing a system-level stochastic model constrained by a large set of single-cell E. coli flagellar synthesis data from different strains and mutants, we identify the general underlying design principles for generating stochastic transcriptional pulses without feedback. Our study shows that an inhibitor (YdiV) of the transcription factor (FlhDC) creates a monotonic ultrasensitive switch that serves as a digital filter to eliminate small-amplitude FlhDC fluctuations. Furthermore, we find that the high-frequency (fast) fluctuations of FlhDC are filtered out by integration over a timescale longer than the timescale of the input fluctuations. Together, our results reveal a filter-and-integrate design for generating stochastic pulses without feedback. This filter-and-integrate mechanism enables a general strategy for cells to avoid premature activation of the expensive downstream gene expression by filtering input fluctuations in both intensity and time so that the system only responds to input signals that are both strong and persistent.

Variables in Cryosurgery Technique Associated With Clearance of Actinic Keratosis.

BACKGROUND: Cryosurgery is the most commonly used method to treat actinic keratosis (AK). Cryosurgical methods are not standardized. OBJECTIVE: To examine differences in the spray techniques used for liquid nitrogen cryosurgery when treating AKs of the head, and the effect of these variations in technique on rates of complete clearance of AKs. MATERIALS AND METHODS: Patients were those from the FIELD-1 study, who received cryosurgery as per the investigators' usual practice to all AKs. This was followed by topical treatment with either vehicle gel or ingenol mebutate gel, 0.015%, after 3 weeks. The investigator recorded the average duration of cryosurgery spray used, the number of freeze-thaw cycles, and the distance from the tip of the spray device to the AK. Clearance rates were determined at Week 11. RESULTS: Less-aggressive freezing techniques were used for AKs on the face than for those on the scalp. However, higher rates of complete clearance on the face and scalp were associated with more-aggressive freezing techniques. CONCLUSION: Patients with AKs on the face receive less-aggressive cryosurgery than do patients with AKs on the scalp.

Regression Analysis of Local Skin Reactions to Predict Clearance of Actinic Keratosis on the Face in Patients Treated With Ingenol Mebutate Gel: Experience from Randomized Controlled Trials.

Ingenol mebutate gel, a topical field treatment for actinic keratosis (AK), elicits inflammatory application-site reactions in most patients. This analysis explored the relationship between the intensity of local skin reactions (LSRs) and AK clearance, measured by the reduction in AK count from baseline in 218 patients who were treated for AK on the face in the pivotal Phase 3 studies. The analysis modeled the AK count at week 8, adjusted for baseline count, with the composite LSR score at 1 day after the last treatment application for each patient as a predictor to estimate the mean and 90% prediction interval for the percent reduction in AK count. The predicted mean percent reduction in AK count was higher in patients with higher composite LSR scores. Lower composite scores demonstrated a variable, less predictive percentage reduction in efficacy. Therefore, a large inflammatory reaction from ingenol mebutate gives a more reliable prognosis for improved AK clearance.

J Drugs Dermatol. 2017;16(2):112-114.

Prevalence of undiagnosed dysglycemia in an emergency department observation unit.

BACKGROUND: The proposed 2015 US Preventive Services Task Force guidelines recommend diabetes screening for individuals ≥45 years or demonstrating other risk factors for dysglycemia. Still, many patients with dysglycemia remain undiagnosed, and opportunities for early intervention are lost. METHODS: To test novel approaches for diagnosis using the haemoglobin A1c (HbA1c ) test, we screened adult patients who were admitted to an observation unit from the emergency department with no known history of pre-diabetes or diabetes. RESULTS: Of 256 subjects, 9% were newly diagnosed with diabetes and 52% were newly diagnosed with pre-diabetes. Of those aged 18-29 years, 33% were newly diagnosed with dysglycemia, while 55% of those aged 30-44 years and 70% of those aged ≥45 years were newly diagnosed with dysglycemia. CONCLUSIONS: Our results suggest that regardless of age, a large proportion of patients in the emergency department observation unit have undiagnosed dysglycemia, an important finding given the large number of observation admissions. Copyright © 2015 John Wiley & Sons, Ltd.

Ingenol mebutate gel for actinic keratosis: the link between quality of life, treatment satisfaction, and clinical outcomes.

BACKGROUND: Actinic keratosis therapy can elicit unsightly and painful local skin responses; assessment of treatment satisfaction and health-related quality of life (QoL) is important. Ingenol mebutate gel is a novel topical field therapy for actinic keratosis. OBJECTIVE: Post-hoc analyses were performed based on patient-reported outcomes from phase-III trials (n = 1005) to assess the effects of ingenol mebutate on QoL and the relationship between both QoL and treatment satisfaction, and degree of lesion clearance. METHODS: Patients received ingenol mebutate or vehicle for self-application to a 25-cm(2) contiguous area: 0.015% once daily for 3 consecutive days (face/scalp) or 0.05% once daily for 2 consecutive days (trunk/extremities). QoL (Skindex-16) and Treatment Satisfaction Questionnaire for Medication data were recorded. RESULTS: Significant, positive associations between Treatment Satisfaction Questionnaire for Medication score and degree of clearance were identified for patients in the face/scalp (effectiveness P < .0001 and global satisfaction P = .0002) and trunk/extremities (P < .0001 and P = .0014, respectively) groups. There was a significant association between Skindex-16 score and clearance for patients in the face/scalp group for change in symptoms (P = .0218), emotions (P = .0002), and overall Skindex-16 score (P = .0006) from baseline. LIMITATIONS: Clinical trial population findings may not be generalizable to clinical practice. CONCLUSION: Ingenol mebutate significantly improved patients' QoL and treatment satisfaction. Improvements were associated with higher degrees of actinic keratosis lesion clearance.

Efficacy and safety of ingenol mebutate 0.015% gel 3 weeks after cryosurgery of actinic keratosis: 11-week results.

INTRODUCTION: Cryosurgery is the most common treatment for actinic keratosis (AK) in the United States. Efficacy with cryosurgery is variable, and is a modality for treating individual, visible lesions while failing to treat subclinical lesions. METHODS: FIELD Study 1 (NCT01541553) is a phase 3, multicenter, randomized, double-blind study that evaluated the short- (11-week) and long- (12-month) term efficacy and safety of sequential AK treatment using cryosurgery with liquid nitrogen followed by ingenol mebutate gel, versus cryosurgery followed by vehicle. RESULTS: Overall, 329 patients were randomized to ingenol mebutate 0.015% gel (n=167) or vehicle (n=162) 3 weeks after cryosurgery. Baseline characteristics were balanced across groups. At week 11, complete clearance rate (100%) in the treatment area was higher for ingenol mebutate gel compared with vehicle (60.5% vs 49.4%, respectively; P=.04). Mean percentage reduction in number of AKs versus baseline was also numerically higher for ingenol mebutate gel (82.7% vs 75.6%). A general reduction from baseline lesion count was observed 3 weeks after cryosurgery. Treatment after cryosurgery was well tolerated. CONCLUSIONS: Short-term (11-week) AK clearance rates on the face or scalp with ingenol mebutate gel after cryosurgery were higher than with cryosurgery alone.

Efficacy and safety of ingenol mebutate 0.015% gel after cryosurgery of actinic keratosis: 12-month results.

INTRODUCTION: Recurrence rates of actinic keratosis (AK) lesions after cryosurgery are high, and this treatment does not address field cancerization. We investigated the efficacy and safety of field treatment of AKs with ingenol mebutate gel following cryosurgery. METHODS: In this phase 3, randomized, double-blind, vehicle-controlled study (NCT01541553), patients ≥18 years with four to eight clinically typical, visible, discrete AKs within a contiguous 25-cm2 treatment area on the face or scalp underwent cryosurgery followed 3 weeks later by once-daily ingenol mebutate 0.015% or vehicle gel for 3 consecutive days. Endpoints included complete clearance at week 11 and safety and efficacy over 12 months. RESULTS: In 329 randomized patients, complete clearance rates were greater with ingenol mebutate than vehicle (week 11: 60.5% vs 49.4%; P=.04; month 12: 30.5% vs 18.5%; P=.01). Fewer patients experienced the emergence of new lesions with ingenol mebutate than with vehicle (38.9% vs 51.9%; P =.02). At month 12, mean percentage reduction of AKs was higher with ingenol mebutate than with vehicle (68.2% vs 54.1%; P =.002). The probability of remaining free of lesions was sustained longer with ingenol mebutate compared with vehicle gel: 78% vs 68% at 6 months; 64% vs 57% at 9 months; 55% vs 40% at month 12, respectively. Ingenol mebutate 0.015% gel was well tolerated and no unexpected adverse events occurred; all adverse events resolved within 2 weeks of starting treatment. CONCLUSIONS: Field treatment with ingenol mebutate 0.015% gel following cryosurgery significantly enhanced clearance of baseline lesions, and was well tolerated. Furthermore, ingenol mebutate 0.015% gel following cryosurgery reduced development of new lesions in the treated field.