Development of an miRFP680-Based Fluorescent Calcium Ion Biosensor Using End-Optimized Transposons.

The development of new or improved single fluorescent protein (FP)-based biosensors (SFPBs), particularly those with excitation and emission at near-infrared wavelengths, is important for the continued advancement of biological imaging applications. In an effort to accelerate the development of new SFPBs, we report modified transposons for the transposase-based creation of libraries of FPs randomly inserted into analyte binding domains, or vice versa. These modified transposons feature ends that are optimized to minimize the length of the linkers that connect the FP to the analyte binding domain. We rationalized that shorter linkers between the domains should result in more effective allosteric coupling between the analyte binding-dependent conformational change in the binding domain and the fluorescence modulation of the chromophore of the FP domain. As a proof of concept, we employed end-modified Mu transposons for the discovery of SFPB prototypes based on the insertion of two circularly permuted red FPs (mApple and FusionRed) into binding proteins for l-lactate and spermidine. Using an analogous approach, we discovered calcium ion (Ca2+)-specific SFPBs by random insertion of calmodulin (CaM)-RS20 into miRFP680, a particularly bright near-infrared (NIR) FP based on a biliverdin (BV)-binding fluorescent protein. Starting from an miRFP680-based Ca2+ biosensor prototype, we performed extensive directed evolution, including under BV-deficient conditions, to create highly optimized biosensors designated the NIR-GECO3 series. We have extensively characterized the NIR-GECO3 series and explored their utility for biological Ca2+ imaging. The methods described in this work will serve to accelerate SFPB development and open avenues for further exploration and optimization of SFPBs across a spectrum of biological applications.

Impact of comorbidities in severely injured patients with blunt chest injury: A population-based retrospective cohort study.

INTRODUCTION: Blunt chest injuries result in up to 10 % of major trauma admissions. Comorbidities can complicate recovery and increase the mortality rate in this patient cohort. A better understanding of the association between comorbidities and patient outcomes will facilitate enhanced models of care for particularly vulnerable groups of patients, such as older adults. AIMS: i) compare the characteristics of severely injured patients with blunt chest injury with and without comorbidities and ii) examine the relationship between comorbidities and key patient outcomes: prolonged length of stay, re-admission within 28 days, and mortality within 30 days in a cohort of patients with blunt chest injury admitted after severe trauma. METHODS: A retrospective cohort study using linked data from the NSW Trauma Registry and NSW mortality and hospitalisation records between 1st of January 2012 and 31st of December 2019. RESULTS: After adjusting for potential confounding factors, patients with severe injuries, chest injuries, and comorbidities were found to have a 34 % increased likelihood of having a prolonged length of stay (OR = 1.34, 95 %I = 1.17-1.53) compared to patients with no comorbidities. There was no difference in 30-day mortality for patients with a severe chest injury who did or did not have comorbidities (OR = 1.05, 95 %CI = 0.80-1.39). No significant association was found between comorbidities and re-admission within 28 days. CONCLUSION: Severely injured patients with blunt chest injury and comorbidities are at risk of prolonged length of stay.

MIR137 polygenic risk for schizophrenia and ephrin-regulated pathway: Role in lateral ventricles and corpus callosum volume.

Background/Objective. Enlarged lateral ventricle (LV) volume and decreased volume in the corpus callosum (CC) are hallmarks of schizophrenia (SZ). We previously showed an inverse correlation between LV and CC volumes in SZ, with global functioning decreasing with increased LV volume. This study investigates the relationship between LV volume, CC abnormalities, and the microRNA MIR137 and its regulated genes in SZ, because of MIR137's essential role in neurodevelopment. Methods. Participants were 1224 SZ probands and 1466 unaffected controls from the GENUS Consortium. Brain MRI scans, genotype, and clinical data were harmonized across cohorts and employed in the analyses. Results. Increased LV volumes and decreased CC central, mid-anterior, and mid-posterior volumes were observed in SZ probands. The MIR137-regulated ephrin pathway was significantly associated with CC:LV ratio, explaining a significant proportion (3.42 %) of CC:LV variance, and more than for LV and CC separately. Other pathways explained variance in either CC or LV, but not both. CC:LV ratio was also positively correlated with Global Assessment of Functioning, supporting previous subsample findings. SNP-based heritability estimates were higher for CC central:LV ratio (0.79) compared to CC or LV separately. Discussion. Our results indicate that the CC:LV ratio is highly heritable, influenced in part by variation in the MIR137-regulated ephrin pathway. Findings suggest that the CC:LV ratio may be a risk indicator in SZ that correlates with global functioning.

Complete genome sequence of Gluconobacter frateurii ML.ISBL3, an endophytic strain isolated from aerial roots of Syngonium podophyllum.

The endophytic strain Gluconobacter frateurii ML.ISBL3 was isolated from aerial roots of Syngonium podophyllum in Hong Kong. Its complete genome, established through hybrid assembly, comprises a single chromosome of 3,309,710 bp (56.30% G+C).

Complete genome sequence of Klebsiella variicola subsp. variicola ML.9ba2, an endophytic strain isolated from aerial roots of Philodendron erubescens.

The endophytic strain Klebsiella variicola subsp. variicola ML.9ba2 was isolated from aerial roots of Philodendron erubescens in Hong Kong. Its complete genome of 5,682,083 bp (57.29% G+C), comprising a single chromosome and an IncF plasmid, was established through hybrid assembly.

The Omicron variant of COVID-19 and its association with croup in children: a single-centre study in Hong Kong.

INTRODUCTION: The fifth wave of the coronavirus disease 2019 (COVID-19) pandemic in Hong Kong was dominated by the Omicron variant, which may cause more upper airway involvement in children. This study was performed to identify any associations between the Omicron variant of COVID-19 and croup in children. METHODS: This retrospective study reviewed the electronic medical records of patients admitted to Tuen Mun Hospital in Hong Kong from 1 January 2018 to 31 March 2022 under the diagnostic code for croup (J05.0 in the International Classification of Diseases 10th Edition). Patients were categorised into three groups according to their admission periods, namely, non-COVID-19, COVID-19-pre-Omicron, and COVID-19-Omicron groups. Disease associations and severity were compared according to incidence, Westley Croup Score, length of hospital stay, medication use, respiratory support, and intensive care unit admissions. RESULTS: The COVID-19 incidence among patients with croup was significantly higher in the COVID-19-Omicron group than in the COVID-19-pre-Omicron group (90.0% vs 2.0%; P<0.001). Compared with patients in the COVID-19-pre-Omicron and non-COVID-19 groups, patients in the COVID-19-Omicron group also had a higher Westley score (moderate and severe disease in the COVID-19-Omicron group: 56.7%; COVID-19-pre-Omicron group: 22.0%, P=0.004; non-COVID-19 group: 24.8%, P<0.001), longer median hospital stay (COVID-19-Omicron group: 3.00 days; COVID-19-pre-Omicron group: 2.00 days, P<0.001; non-COVID-19 group: 2.00 days, P=0.034), and higher mean dexamethasone requirement (COVID-19-Omicron group: 0.78 mg/kg; COVID-19-pre-Omicron group: 0.49 mg/kg, P<0.001; non-COVID-19 group: 0.58 mg/kg, P=0.001). CONCLUSION: The Omicron variant of COVID-19 is associated with croup and can cause more severe disease in Hong Kong children.

Dose-effect relationships in neuroendocrine tumour liver metastases treated with [166Ho]-radioembolization.

PURPOSE: Aim of this study was to investigate a dose-response relationship, dose-toxicity relationship, progression free survival (PFS) and overall survival (OS) in neuroendocrine tumour liver metastases (NELM) treated with holmium-166-microspheres radioembolization ([166Ho]-radioembolization). MATERIALS AND METHODS: Single center, retrospective study included patients with NELM that received [166Ho]-radioembolization with post-treatment SPECT/CT and CECT or MRI imaging for 3 months follow-up. Post-treatment SPECT/CT was used to calculate tumour (Dt) and whole liver healthy tissue (Dh) absorbed dose. Clinical and laboratory toxicity was graded by Common Terminology Criteria for Adverse Events (CTCAE), version 5 at baseline and three-months follow-up. Response was determined according to RECIST 1.1. The tumour and healthy doses was correlated to lesion-based objective response and patient-based toxicity. Kaplan Meier analyses were performed for progression free survival (PFS) and overall survival (OS). RESULTS: Twenty-seven treatments in 25 patients were included, with a total of 114 tumours. Median follow-up was 14 months (3 - 82 months). Mean Dt in non-responders was 68 Gy versus 118 Gy in responders, p = 0.01. ROC analysis determined 86 Gy to have the highest sensitivity and specificity, resp. 83% and 81%. Achieving a Dt of ≥ 120 Gy provided the highest likelihood of response (90%) for obtaining response. Sixteen patients had grade 1-2 clinical toxicity and only one patient grade 3. No clear healthy liver dose-toxicity relationship was found. The median PFS was 15 months (95% CI [10.2;19.8]) and median OS was not reached. CONCLUSION: This study confirms the safety and efficacy of [166Ho]-radioembolization in NELM in a real-world setting. A clear dose-response relationship was demonstrated and future studies should aim at a Dt of ≥ 120 Gy, being predictive of response. No dose-toxicity relationship could be established.

Intra-arterial peptide-receptor radionuclide therapy for neuro-endocrine tumour liver metastases: an in-patient randomised controlled trial (LUTIA).

PURPOSE: Peptide receptor radionuclide therapy (PRRT) using [177Lu]Lu-DOTATATE has been shown to effectively prolong progression free survival in grade 1-2 gastroenteropancreatic neuroendocrine tumours (GEP-NET), but is less efficacious in patients with extensive liver metastases. The aim was to investigate whether tumour uptake in liver metastases can be enhanced by intra-arterial administration of [177Lu]Lu-DOTATATE into the hepatic artery, in order to improve tumour response without increasing toxicity. METHODS: Twenty-seven patients with grade 1-2 GEP-NET, and bi-lobar liver metastases were randomized to receive intra-arterial PRRT in the left or right liver lobe for four consecutive cycles. The contralateral liver lobe and extrahepatic disease were treated via a "second-pass" effect and the contralateral lobe was used as the control lobe. Up to three metastases (> 3 cm) per liver lobe were identified as target lesions at baseline on contrast-enhanced CT. The primary endpoint was the tumour-to-non-tumour (T/N) uptake ratio on the 24 h post-treatment [177Lu]Lu-SPECT/CT after the first cycle. This was calculated for each target lesion in both lobes using the mean uptake. T/N ratios in both lobes were compared using paired-samples t-test. FINDINGS: After the first cycle, a non-significant difference in T/N uptake ratio was observed: T/NIA = 17·4 vs. T/Ncontrol = 16·2 (p = 0·299). The mean increase in T/N was 17% (1·17; 95% CI [1·00; 1·37]). Of all patients, 67% (18/27) showed any increase in T/N ratio after the first cycle. CONCLUSION: Intra-arterial [177Lu]Lu-DOTATATE is safe, but does not lead to a clinically significant increase in tumour uptake.

Effect of Community Treatment Orders on Mental Health Service Usage, Emergency Visits, and Violence: a Systematic Review and Meta-Analysis.

INTRODUCTION: Community treatment orders (CTOs) enable patients to actively engage in mental health services while being supervised in the community outside the hospital setting. However, the efficacy of CTOs remains controversial in terms of mental health services usage or service contacts, emergency visits, and violence. METHODS: The databases PsychINFO, Embase, and Medline were searched on 11 March 2022 by 2 independent reviewers through the Covidence website (www.covidence.org). Randomised or non-randomised case-control studies and pre-post studies were included if they examine the effect of CTOs on service contacts, emergency visits, and violence in individuals with mental illnesses by comparing with control groups or pre-CTO conditions. Conflicts were resolved by consultation of the third independent reviewer. RESULTS: Sixteen studies provided sufficient data in the target outcome measures and were included in analysis. Variability in the risk of bias was high among studies. Meta-analyses were conducted separately for case-control studies and pre-post studies. For service contacts, a total of 11 studies with 66,192 patients reported changes in the number of service contacts under CTOs. In 6 case-control studies, a small non-significant increase in service contacts was observed in those under CTOs (Hedge's g = 0.241, z = 1.535, p = 0.13). In 5 pre-post studies, a large and significant increase in service contacts was noted after CTOs (Hedge's g = 0.830, z = 5.056, p < 0.001). For emergency visits, a total of 6 studies with 930 patients reported changes in the number of emergency visits under CTOs. In 2 case-control studies, a small non-significant increase in emergency visits was noted in those under CTOs (Hedge's g = -0.196, z = -1.567, p = 0.117). In 4 pre-post studies, a small significant decrease in emergency visits was noted after CTOs (Hedge's g = 0.553, z = 3.101, p = 0.002). For violence, a total of 2 pre-post studies reported a moderate significant reduction in violence after CTOs (Hedge's g = 0.482, z = 5.173, p < 0.001). CONCLUSION: Case-control studies showed inconclusive evidence, but pre-post studies showed significant effects of CTOs in promoting service contacts and reducing emergency visits and violence. Future studies on cost-effectiveness analysis and qualitative analysis for specific populations with various cultures and backgrounds are warranted.

Application of virtual reality for peritoneal dialysis exchange learning in patients with end-stage renal disease and cognitive impairment.

Cognitive impairment is not uncommon in patients with end-stage renal disease and can make it more difficult for these patients to carry out peritoneal dialysis (PD) on their own. Their attempts to do so may result in adverse consequences such as peritonitis. PD exchange is a complex procedure demanding knowledge and skill which requires close supervision and guidance by a renal nurse specialist. In this study, a non-immersive virtual reality (VR) training program using a Leap motion hand tracking device was developed to facilitate patients' understanding and learning of the PD exchange procedure before attempting real task practice. This study was a two-center single-blinded randomized controlled trial on 23 incident PD patients. Patients in the experimental group received 8 sessions of VR training, while patients in the control were provided with printed educational materials. The results showed that there were significant differences between the two groups in performance of the overall PD exchange sequence, especially on the crucial steps. VR had a patient satisfaction rate of 89%, and all patients preferred to have the VR aid incorporated in PD training. Our findings conclude VR can be a useful aid in the training and reinforcement of PD exchange procedures, with distinct merits of being free from restrictions of time, space, and manpower.

Virtual patient-specific QA with DVH-based metrics.

We demonstrate a virtual pretreatment patient-specific QA (PSQA) procedure that is capable of quantifying dosimetric effect on patient anatomy for both intensity modulated radiotherapy (IMRT) and volumetric modulated arc therapy (VMAT). A machine learning prediction model was developed to use linear accelerator parameters derived from the DICOM-RT plan to predict delivery discrepancies at treatment delivery (defined as the difference between trajectory log file and DICOM-RT) and was coupled with an independent Monte Carlo dose calculation algorithm for dosimetric analysis. Machine learning models for IMRT and VMAT were trained and validated using 120 IMRT and 206 VMAT fields of prior patients, with 80% assigned for iterative training and testing, and 20% for post-training validation. Various prediction models were trained and validated, with the final models selected for clinical implementation being a boosted tree and bagged tree for IMRT and VMAT, respectively. After validation, these models were then applied clinically to predict the machine parameters at treatment delivery for 7 IMRT plans from various sites (61 fields) and 10 VMAT multi-target intracranial radiosurgery plans (35 arcs) and compared to the dosimetric effect calculated directly from trajectory log files. Dose indices tracked for targets and organs at risk included dose received by 99%, 95%, and 1% of the volume, mean dose, percent of volume receiving 25%-100% of the prescription dose. The average coefficient of determination (r2 ) when comparing intra-field predicted and actual delivery error was 0.987 ± 0.012 for IMRT and 0.895 ± 0.095 for VMAT, whereas r2 when comparing inter-field predicted versus actual delivery error was 0.982 for IMRT and 0.989 for VMAT. Regarding dosimetric analysis, r2 when comparing predicted versus actual dosimetric changes for all dose indices was 0.966 for IMRT and 0.907 for VMAT. Prediction models can be used to anticipate the dosimetric effect calculated from trajectory files and have potential as a "delivery-free" pretreatment analysis to enhance PSQA.

Challenges in Von Hippel-Lindau's disease: PRRT in patients on hemodialysis.

Summary: Von Hippel-Lindau's disease (VHL) is a hereditary tumor syndrome characterized by its prototype lesions, hemangioblastomas, and renal cell carcinomas. Treatment for renal cell carcinomas can ultimately result in long-term dialysis. Pancreatic neuroendocrine tumors (pNET) can also occur in the course of the disease. Currently, peptide receptor radionuclide therapy (PRRT) is the standard treatment for progressive neuroendocrine tumors. However, little is known about treatment with PRRT in patients on dialysis, an infrequent presentation in patients with VHL. We present a 72-year-old man with VHL on hemodialysis and a progressive pNET. He received four cycles of PRRT with a reduced dose. Only mild thrombopenia was seen during treatments. The patient died 9 months after the last PRRT because of acute bleeding in a hemangioblastoma. Hemodialysis is not a limiting factor for PRRT treatment and it should be considered as it seems a safe short-term treatment option for this specific group. Learning points: Von Hippel-Lindau disease (VHL) is a complex disease in which former interventions can limit optimal treatment for following VHL-related tumors later in life. Metastasized pancreatic neuroendocrine tumors occur as part of VHL disease. Peptide receptor radionuclide therapy seems a safe short-term treatment option in patients on hemodialysis.

Characterization of plasmatic proteins adsorption on poly(styrene sodium sulfonate) functionalized silicone surfaces.

Proteins adsorption occurs spontaneously on biomaterial upon insertion within the body. The resulting protein layer influences biomaterial biocompatibility through enhanced bio-integration or, on the contrary, adverse reactions. Furthermore, upon adsorption, proteins can undergo modifications of their structure and, ultimately, their physicochemical properties and activity. Hence, the understanding of protein adsorption on implanted materials appears essential, as exemplified by silicone breast prostheses that might lead to serious health issues. Surface modifications with a bioactive polymer, poly(styrene sodium sulfonate)-polyNaSS, on a hydrophobic silicone surface that composes breast implants, have been successfully performed under UV irradiation by a radical surface polymerization. This strategy enhances cell biocompatibility and antibacterial features. Although detailed insights related to the mechanism are still scarce, polyNaSS is supposed to promote changes in the conformation and/or orientation of adsorbed plasma proteins, reducing the odd for a biofilm to form. The present work addresses more in-depth structural investigations of the adsorbed state of two plasma proteins: Bovine Serum Albumin (BSA), as a model protein, and fibronectin (FN), for its role in cell adhesion. Using Atomic force microscopy (AFM), we report that polyNaSS showed no significant impact on the BSA structure conversely to the FN one. However, imaging findings with AFM clearly outlined a change in the structural organization of FN, going from a nano fibrillar assembly with an average length of 130 nm to a globular one when the surface was grafted. Thus, it is highlighted that polyNaSS interacts specifically with FN. In addition, cell spreading assay of L929 fibroblasts on FN-coated surfaces with optical microscopy indicated no significant impact of the change in FN structure upon fibroblasts adhesion, which displayed active elongated shapes. The present features are crucial for understanding the cell adhesion mechanism induced by surface modification.

EANM procedure guideline for the treatment of liver cancer and liver metastases with intra-arterial radioactive compounds.

Primary liver tumours (i.e. hepatocellular carcinoma (HCC) or intrahepatic cholangiocarcinoma (ICC)) are among the most frequent cancers worldwide. However, only 10-20% of patients are amenable to curative treatment, such as resection or transplant. Liver metastases are most frequently caused by colorectal cancer, which accounts for the second most cancer-related deaths in Europe. In both primary and secondary tumours, radioembolization has been shown to be a safe and effective treatment option. The vast potential of personalized dosimetry has also been shown, resulting in markedly increased response rates and overall survival. In a rapidly evolving therapeutic landscape, the role of radioembolization will be subject to changes. Therefore, the decision for radioembolization should be taken by a multidisciplinary tumour board in accordance with the current clinical guidelines. The purpose of this procedure guideline is to assist the nuclear medicine physician in treating and managing patients undergoing radioembolization treatment. PREAMBLE: The European Association of Nuclear Medicine (EANM) is a professional non-profit medical association that facilitates communication worldwide among individuals pursuing clinical and research excellence in nuclear medicine. The EANM was founded in 1985. These guidelines are intended to assist practitioners in providing appropriate nuclear medicine care for patients. They are not inflexible rules or requirements of practice and are not intended, nor should they be used, to establish a legal standard of care. The ultimate judgment regarding the propriety of any specific procedure or course of action must be made by medical professionals taking into account the unique circumstances of each case. Thus, there is no implication that an approach differing from the guidelines, standing alone, is below the standard of care. To the contrary, a conscientious practitioner may responsibly adopt a course of action different from that set out in the guidelines when, in the reasonable judgment of the practitioner, such course of action is indicated by the condition of the patient, limitations of available resources or advances in knowledge or technology subsequent to publication of the guidelines. The practice of medicine involves not only the science but also the art of dealing with the prevention, diagnosis, alleviation and treatment of disease. The variety and complexity of human conditions make it impossible to always reach the most appropriate diagnosis or to predict with certainty a particular response to treatment. Therefore, it should be recognised that adherence to these guidelines will not ensure an accurate diagnosis or a successful outcome. All that should be expected is that the practitioner will follow a reasonable course of action based on current knowledge, available resources and the needs of the patient to deliver effective and safe medical care. The sole purpose of these guidelines is to assist practitioners in achieving this objective.

Narrow pH tolerance found for a microbial fuel cell treating winery wastewater.

AIMS: The use of microbial fuel cells (MFC) to treat winery wastewater is promising; however, an initial acidic pH, fluctuating chemical oxygen demand (COD) levels and a lack of natural buffering in these wastewaters make providing a suitable buffer system at an ideal buffer to COD ratio. METHODS AND RESULTS: A lab scale MFC was designed, inoculated with anaerobic winery sludge and fed with synthetic winery wastewater. It was observed that at pH 6·5, the MFC performed best, the maximum output voltage was 0·63 ± 0·01 V for 60 ± 3 h, and the COD removal efficiency reached 77 ± 7%. The electrogens were affected by pH much more than the bulk COD degrading organisms. Fluorescent in situ hybridization suggested Betaproteobacteria played a significant role in electron transfer. CONCLUSIONS: A ratio of 1 mmol l-1 phosphate buffer to 100 mg l-1 COD was ideal to maintain a stable pH for MFCs treating synthetic winery wastewater. SIGNIFICANCE AND IMPACT OF THE STUDY: The results find the narrow pH tolerance for MFCs treating winery wastewater and demonstrate the significance of pH and buffer to COD ratio for steady performance of MFCs.

A questionnaire survey on patients' willingness to pay with reference to the waiting time of public in-vitro fertilization treatment in Hong Kong.

OBJECTIVE: To evaluate patients' willingness to pay (WTP) with reference to the waiting time of public in-vitro fertilisation (IVF) treatment in order to improve the public IVF service in Hong Kong. STUDY DESIGN: A prospective multi-centred questionnaire survey. Infertile women attending infertility clinics of nine public hospitals in Hong Kong between October 2017 and August 2018 were asked to complete a questionnaire in their first clinic visit. RESULTS: Out of 1092 respondents, 10.4 % had private IVF cycles prior to their first visit at public hospitals. In general, patients were willing to pay more for a shorter waiting time for public IVF service. The proportion of respondents who were willing to pay more than HK$10,000 (US$1282) for one IVF cycle increased from 54.6% to 80.7% if the waiting time for public IVF service were hypothetically shortened from four years to one year. Likewise, 22.5 % versus 45.5 % were willing to pay more than HK$ 25,000 (US$3205) with a waiting time of four versus one year respectively. Assuming the cost per IVF cycle was HK$ 25,000 (US$3205), 23.4 % of respondents could afford one IVF cycle, 40.0 % of them could afford two IVF cycles and 31.5 % could afford three IVF cycles. A multivariate regression model demonstrated that only family income and presence of existing child(ren) were significant independent determinants of the maximum amount that an individual was willing to pay for IVF (p < 0.05). Those with family monthly income below HK$100,000 ($12,820) were less than half as likely, and those without existing child(ren) were more than double as likely, to be willing to pay higher for IVF. CONCLUSION: Patients were willing to pay more for a shorter waiting time for public IVF service. Those with family income below HK$100,000 (US$ 12,820) were less than half as likely, and those without existing children were more than double as likely, to be willing to pay higher for IVF.