Adaptive radiotherapy of locally advanced sigmoid colon cancer with intra­fractional motion using the MRIdian system: A case report.

Neoadjuvant chemotherapy, when combined with radiotherapy, serves as an optional treatment for patients with locally advanced sigmoid colon cancer and is usually performed in conjunction with complete mesocolic excision. The substantial movement of surrounding organs in cases of sigmoid colon cancer frequently leads to toxicity in normal tissues. The present report details the case of a 76-year-old man diagnosed with locally advanced sigmoid colon cancer. Initially, treatment using the Tomotherapy Hi-Art system was selected; however, during image guidance from the first to the sixth fractions, the tumor location underwent a marked change, exceeding the range of the planning target volume. Efforts to recapture the image were unsuccessful, leading to a decision to transition the patient to the MRIdian system for daily treatment with online adaptive radiotherapy. The positional variations in the tumor were evident in each treatment using the MRIdian system, with mean shifts of 2.58 cm in the right-left direction, 1.24 cm in the cranial-caudal direction and 0.40 cm in the anterior-posterior direction. The mean time from the entry of the patient to treatment completion was 41 min. Adaptive treatment plans were performed for all 19 fractions, with two treatments repeated due to the tumor moving out of tracking range. Following irradiation using the MRIdian system, the gross tumor volume decreased by 62%. Notably, the patient experienced no side effects during treatment. A CT scan conducted 3 months after radiotherapy revealed a marked reduction in the tumor size, consistent with a partial response, leading to the scheduling of surgery. Following surgery, a CT scan after 6 months revealed no local recurrence in the surgical bed region. The findings in the present case support the feasibility of implementing an adaptive treatment plan using the MRIdian system for locally advanced sigmoid colon cancer in the context of neoadjuvant chemoradiotherapy.

The impact of place attachment on well-being for older people in high-density urban environment: a qualitative study.

Aging in place has become a popular social policy worldwide. This paper argues that well-being is an important outcome of aging in place, upon which older people develop autonomy and environmental proactivity. The temporal dimension of aging in place highlights development of place attachment, which includes place identity and place dependence. The study explores how older people, who live inhigh-density urban environments, make sense of well-being and place attachment by articulating their daily lives. Community dwelling older people aged 65 and above, who came from neighborhoods with high aging population and residential density but high and low median household incomes, were invited for focus group discussions. Multifaceted meanings of well-being include various dimensions that cover individual-collective and material-spiritual (psychological) construct. Meanings of place attachment include values of, bonding ties to, and memories about places. Three pathways are identified linking place attachment and multifaceted well-being. The study finds that social welfare and material richness are not the only determinants of well-being. Fulfillment of higher psychological needs, such as positive evaluation of life and self-actualization, should be emphasized by which older people can make the most of their life in old age.

Case report and literature review: Conversion surgery for initially unresectable huge retroperitoneal liposarcoma after preoperative radiotherapy.

Background: Retroperitoneal liposarcoma (RPLS) is a rare malignancy that is notorious for recurrence. Surgical resection with clean margin is the current treatment of choice. However, owing to the large retroperitoneal space, RPLSs often grow to significant sizes before being diagnosed. Neoadjuvant and adjuvant therapies have potentials to improve long term treatment outcome. Case presentation: A 55-year-old Han Chinese male presented to the general surgery department with a one-year history of abdominal fullness and a one-week history of palpable right inguinal mass. At first, he was diagnosed with incarcerated inguinal hernia. However, abdominal computer tomography (CT) and biopsy confirmed his final diagnosis to be retroperitoneal well-differentiated liposarcoma, cT2bN0M0, stage IIb. The tumor, which measured 44.5cm in maximum diameter, was too large for primary surgical resection. Neoadjuvant radiotherapy with 70 Gy in 35 fractions was delivered to the tumor, which shrunk the target volume from 6300 cc to 4800 cc, as observed in the middle of the radiotherapy course. The right testicular mass also received 70Gy/35Fx. Conversion surgery was performed after radiotherapy. Unfortunately, due to residual tumor, adjuvant chemotherapy consisting of AIM (ifosfamide, Mesna, and doxorubicin) and MAID (Mesna, doxorubincin, ifosfamide, and dacarbazine) regimens were administered sequentially. Afterward, debulking surgery was conducted, plus another 18 cycles of ifosfamide monotherapy when residual tumor was still seen on CT. Since the completion of ifosfamide chemotherapy, the patient has been cancer free with no evidence of tumor recurrence for more than 26 months. Conclusion: Despite conflicting evidence in the literature, our case supports the use of high dose neoadjuvant radiotherapy and adjuvant chemotherapy in treating large, unresectable RPLSs. It also highlights the importance of using individualized, multidisciplinary approach in achieving cure for large, unresectable rare tumors.

Maintaining Quality of Care among Dialysis Patients in Affected Areas after Typhoon Morakot.

Natural disasters have negative health impacts on patients who need dialysis in affected areas. Severely affected areas are usually rural, with limited basic infrastructure and a population without optimal dialysis-specific care after a disaster. A population-based longitudinal case-cohort study enrolled 715,244 adults from the National Health Insurance Registry who lived in areas affected by a major natural disaster, Typhoon Morakot, in 2009. The observation period was from 2008 to 2011. A total of 13,268 patients (1.85%) had a history of end-stage renal disease (ESRD). Of the ESRD patients, 1264 patients (9.5%) received regular dialysis. Only eight patients missed dialysis sessions in the first month after the disaster. Compared to the moderately affected areas, the incidences of acute cerebrovascular and cardiovascular diseases were higher in patients in severely affected areas. Male dialysis patients aged 45-75 years had a higher mortality rate than that of the general population. Among the affected adults receiving regular dialysis, patients with diabetes (adjusted hazard ratio (aHR): 1.58, 95% confidence interval (CI): 1.20-2.08) or a history of cerebrovascular disease (aHR: 1.58, 95% CI: 1.12-2.21), chronic obstructive pulmonary disease (COPD) or asthma (aHR: 1.99, 95% CI: 1.24-3.17) in moderately affected areas had significantly elevated mortality rates. Additionally, among dialysis patients living in severely affected areas, those with a history of cerebrovascular disease (aHR: 4.52 95% CI: 2.28-8.79) had an elevated mortality rate. Early evacuation plans and high-quality, accessible care for cardiovascular and cerebrovascular diseases are essential to support affected populations before and after disasters to improve dialysis patients' health outcomes.

Increased incidence of stress-associated illnesses among elderly after Typhoon Morakot.

BACKGROUND/PURPOSE: Typhoon Morakot in August 2009 caused significant damages and health and socio-economic impacts in Taiwan. Therefore, we evaluated the mental health status of adult and patients who lived in the affected area after the disaster. METHODS: An observational, prospective population-based study was conducted. Adults living in the affected area were selected as the affected population in the National Health Insurance Database from January 2008 to December 2011. Prevalence and incidence of stress-associated illnesses, such as insomnia, anxiety, depressive, adjustment and mood disorders, post-traumatic stress disorder (PTSD) in the psychiatry department were analysed after the disaster. RESULTS: A total of 897,689 adult patients were studied. Of the affected population without pre-existing chronic mental health illness, the monthly visits for stress-associated illnesses, such as insomnia, anxiety, depressive disorders and PTSD increased about twice after the disaster in elderly and non-elderly groups. Comparing to the non-elderly group, the elderly group has more increased in the incidence of insomnia (356% vs. 318% increase) and depressive disorders (308% vs. 245%) but was affected to a lesser extent increase in the anxiety (269% vs. 307%), PTSD, episodic mood disorders (82% vs. 158%), and adjustment reaction (160% vs. 202%). CONCLUSION: The mental health statuses of patients who experienced a major natural disaster deteriorated in the elderly population after the disaster. However, we still need pay more attentions on the elderly of the affected population to decrease the risk for insomnia and depressive disorders after the disaster.

Increased Medical Visits and Mortality among Adults with Cardiovascular Diseases in Severely Affected Areas after Typhoon Morakot.

Natural disasters have negative health impacts on chronic diseases in affected populations. Severely affected areas are usually rural areas with limited basic infrastructure and a population have that has limited access to optimal healthcare after a disaster. Patients with cardiovascular diseases are required to maintain quality care, especially after disasters. A population-based case-control study enrolled adults from the National Health Insurance Registry who had ischemic heart disease and cerebrovascular disease histories and lived in the area affected by Typhoon Morakot in 2009. Monthly medical visits for acute cerebrovascular and ischemic heart diseases markedly increased at approximately 1-2 months after the typhoon. Survival analysis during the two years following the typhoon indicated a significant increase in mortality in adults with an acute ischemic heart disease history who lived in the severely affected area. Mortality hazard analysis showed that among affected adults with previous cerebrovascular diseases and acute ischemic heart diseases, patients with diabetes (adjusted hazard ratio [HR]: 1.3-1.7), Chronic Kidney Disease (CKD) (adjusted HR: 2.0-2.7), chronic obstructive pulmonary diseases (COPD) and asthma (adjusted HR: 1.7-2.1), liver cirrhosis (adjusted HR: 2.3-3.3) and neoplasms (adjusted HR: 1.1-2.1) had significantly increased mortality rates. Consequently, high-quality and accessible primary healthcare plans should be made available to maintain and support affected populations after disasters.

In Vivo Efficacy of SYN023, an Anti-Rabies Monoclonal Antibody Cocktail, in Post-Exposure Prophylaxis Animal Models.

Rabies immune globulin (RIG) is an indispensable component of rabies post-exposure prophylaxis (PEP) because it provides passive immunity to prevent this otherwise inescapably fatal disease in Category III exposed patients. Even with decades of development, RIG products are still criticized for their high cost, lot-to-lot variation, and potential safety issues. They remain largely unattainable in most developing regions of the world, where demand is highest. In recent years, monoclonal antibodies (MAbs) have become widely accepted as safer and more cost-effective alternatives to RIG products. As an example, SYN023 is a 1:1 cocktail of two humanized anti-rabies MAbs previously shown to display extensive neutralizing capabilities. Here, we further assessed the efficacy of SYN023 in animal models of rabies, and found that SYN023 afforded protection equal to a standard dose of human RIG (HRIG) at 0.03 mg/kg in Syrian hamsters and 0.1 mg/kg in beagles. Potential interference with vaccine-induced immunity was analyzed for the MAbs at these concentrations. While individual MAbs did not interfere with vaccine response, SYN023 at dosages of 0.1 mg/kg and above resulted in reduced neutralizing antibody titers similar to HRIG. Thus, the in vivo characterization of SYN023 supports its utility in human rabies PEP as an efficacious alternative to RIG products.

SYN023, a novel humanized monoclonal antibody cocktail, for post-exposure prophylaxis of rabies.

Rabies is a neglected zoonotic disease that is preventable in humans by appropriate post-exposure prophylaxis (PEP). However, current PEP relies on polyclonal immune globulin products purified from pooled human (HRIG) or equine (ERIG) plasma that are either in chronic shortage or in association with safety concerns. Here, we present the development of an antibody cocktail, SYN023, made of two novel monoclonal antibodies (MAb) CTB011 and CTB012 that could serve as safer and more cost-effective alternatives to the current RIG products. Both CTB011 and CTB012 are humanized MAbs that bind to non-overlapping epitopes on the rabies virus (RABV) glycoprotein (G) with sub-nanomolar affinities. Sequence analysis revealed that many of the critical residues in binding are highly conserved across different species of lyssaviruses. When combined at a 1:1 ratio, CTB011/CTB012 exhibited neutralization capabilities equivalent or superior to HRIG against 10 North American street RABV isolates in vitro and 15 prevalent Chinese RABV strains in animal models. Finally, SYN023, at a dosage of 0.03 mg/kg, was able to offer the same degree of protection as standard HRIG administration (20 IU/kg) in Syrian hamsters challenged with a highly virulent bat (Tadarida brasiliensis) RABV variant. Taken together, the high-potency and broad-spectrum neutralization demonstrated by SYN023 make it an effective candidate for human rabies PEP consideration.

Construction of a versatile expression library for all human single-pass transmembrane proteins for receptor pairings by high throughput screening.

Interactions between protein ligands and receptors play crucial roles in cell-cell signalling. Most of the human cell surface receptors have been identified in the post-Human Genome Project era but many of their corresponding ligands remain unknown. To facilitate the pairing of orphan receptors, 2762 sequences encoding all human single-pass transmembrane proteins were selected for inclusion into a mammalian-cell expression library. This expression library, consisting of all the individual extracellular domains (ECDs), was constructed as a Fab fusion for each protein. In this format, individual ECD can be produced as a soluble protein or displayed on cell surface, depending on the applied heavy-chain Fab configuration. The unique design of the Fab fusion concept used in the library led to not only superior success rate of protein production, but also versatile applications in various high-throughput screening paradigms including protein-protein binding assays as well as cell binding assays, which were not possible for any other existing expression libraries. The protein library was screened against human coagulation factor VIIa (FVIIa), an approved therapeutic for the treatment of hemophilia, for binding partners by AlphaScreen and ForteBio assays. Two previously known physiological ligands of FVIIa, tissue factor (TF) and endothelial protein C receptor (EPCR) were identified by both assays. The cell surface displayed library was screened against V-domain Ig suppressor of T-cell activation (VISTA), an important immune-checkpoint regulator. Immunoglobulin superfamily member 11 (IgSF11), a potential target for cancer immunotherapy, was identified as a new and previously undescribed binding partner for VISTA. The specificity of the binding was confirmed and validated by both fluorescence-activated cell sorting (FACS) and surface plasmon resonance (SPR) assays in different experimental setups.

Human Cancer Antigen Globo H Is a Cell-Surface Ligand for Human Ribonuclease 1.

Pancreatic-type ribonucleases are secretory enzymes that catalyze the cleavage of RNA. Recent efforts have endowed the homologues from cow (RNase A) and human (RNase 1) with toxicity for cancer cells, leading to a clinical trial. The basis for the selective toxicity of ribonuclease variants for cancerous versus noncancerous cells has, however, been unclear. A screen for RNase A ligands in an array of mammalian cell-surface glycans revealed strong affinity for a hexasaccharide, Globo H, that is a tumor-associated antigen and the basis for a vaccine in clinical trials. The affinity of RNase A and RNase 1 for immobilized Globo H is in the low micromolar-high nanomolar range. Moreover, reducing the display of Globo H on the surface of human breast adenocarcinoma cells with a small-molecule inhibitor of biosynthesis or a monoclonal antibody antagonist decreases the toxicity of an RNase 1 variant. Finally, heteronuclear single quantum coherence (HSQC) NMR spectroscopy showed that RNase 1 interacts with Globo H by using residues that are distal from the enzymic active site. The discovery that a systemic human ribonuclease binds to a moiety displayed on human cancer cells links two clinical paradigms and suggests a mechanism for innate resistance to cancer.

Fluorogenic label to quantify the cytosolic delivery of macromolecules.

The delivery of a macromolecule to the cytosol of human cells is assessed by using a pendant di-O-glycosylated derivative of fluorescein. Its fluorescence is unmasked by Escherichia coliß-galactosidase installed in the cytosol. Background is diminished by using RNAi to suppress the expression of GLB1, which encodes a lysosomal ß-galactosidase. This strategy was used to quantify the cytosolic entry of a highly cationic protein, ribonuclease A.

Mechanism of ribonuclease A endocytosis: analogies to cell-penetrating peptides.

Pancreatic-type ribonucleases can exert toxic activity by catalyzing the degradation of cellular RNA. Their ability to enter cells is essential for their cytotoxicity. Here, we determine the mechanism by which bovine pancreatic ribonuclease (RNase A) enters human cells. Inhibiting clathrin-dependent endocytosis with dynasore or chlorpromazine decreases RNase A-uptake by ~70%. Limited colocalization between RNase A and transferrin indicates that RNase A is not routed through recycling endosomes. Instead, vesicular staining of RNase A overlaps substantially with that of nona-arginine and the cationic peptide corresponding to residues 47-57 of the HIV-1 TAT protein. At low concentrations (<5 µM), internalization of RNase A and these cell-penetrating peptides (CPPs) is inhibited by chlorpromazine as well as the macropinocytosis inhibitors cytochalasin D and 5-(N-ethyl-N-isopropyl)amiloride to a similar extent, indicative of common endocytic mechanism. At high concentrations, CPPs adopt a nonendocytic mechanism of cellular entry that is not shared by RNase A. Collectively, these data suggest that RNase A is internalized via a multipathway mechanism that involves both clathrin-coated vesicles and macropinosomes. The parallel between the uptake of RNase A and CPPs validates reference to RNase A as a "cell-penetrating protein".

Synthesis and utility of fluorogenic acetoxymethyl ethers.

Phenolic fluorophores such as fluorescein, Tokyo Green, resorufin, and their derivatives are workhorses of biological science. Acylating the phenolic hydroxyl group(s) in these fluorophores masks their fluorescence. The ensuing ester is a substrate for cellular esterases, which can restore fluorescence. These esters are, however, notoriously unstable to hydrolysis, severely compromising their utility. The acetoxymethyl (AM) group is an esterase-sensitive motif that can mask polar functionalities in small molecules. Here, we report on the use of AM ether groups to mask phenolic fluorophores. The resulting profluorophores have a desirable combination of low background fluorescence, high chemical stability, and high enzymatic reactivity, both in vitro and in cellulo. These simple phenyl ether-based profluorophores could supplement or supplant the use of phenyl esters for imaging biochemical and biological systems.

Cellular uptake of ribonuclease A relies on anionic glycans.

Bovine pancreatic ribonuclease (RNase A) can enter human cells, even though it lacks a cognate cell-surface receptor protein. Here, we report on the biochemical basis for its cellular uptake. Analyses in vitro and in cellulo revealed that RNase A interacts tightly with abundant cell-surface proteoglycans containing glycosaminoglycans, such as heparan sulfate and chondroitin sulfate, as well as with sialic acid-containing glycoproteins. The uptake of RNase A correlates with cell anionicity, as quantified by measuring electrophoretic mobility. The cellular binding and uptake of RNase A contrast with those of Onconase, an amphibian homologue that does not interact tightly with anionic cell-surface glycans. As anionic glycans are especially abundant on human tumor cells, our data predicate utility for mammalian ribonucleases as cancer chemotherapeutic agents.

A highly sensitive fluorogenic probe for cytochrome P450 activity in live cells.

A derivative of rhodamine 110 has been designed and assessed as a probe for cytochrome P450 activity. This probe is the first to utilize a 'trimethyl lock' that is triggered by cleavage of an ether bond. In vitro, fluorescence was manifested by the CYP1A1 isozyme with k(cat)/K(M)=8.8x10(3)M(-1)s(-1) and K(M)=0.09microM. In cellulo, the probe revealed the induction of cytochrome P450 activity by the carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin, and its repression by the chemoprotectant resveratrol.

Cytotoxic ribonucleases: the dichotomy of Coulombic forces.

Cells tightly regulate their contents. Still, nonspecific Coulombic interactions between cationic molecules and anionic membrane components can lead to adventitious endocytosis. Here, we characterize this process in a natural system. To do so, we create variants of human pancreatic ribonuclease (RNase 1) that differ in net molecular charge. By conjugating a small-molecule latent fluorophore to these variants and using flow cytometry, we are able to determine the kinetic mechanism for RNase 1 internalization into live human cells. We find that internalization increases with solution concentration and is not saturable. Internalization also increases with time to a steady-state level, which varies linearly with molecular charge. In contrast, the rate constant for internalization (t1/2 = 2 h) is independent of charge. We conclude that internalization involves an extracellular equilibrium complex between the cationic proteins and abundant anionic cell-surface molecules, followed by rate-limiting internalization. The enhanced internalization of more cationic variants of RNase 1 is, however, countered by their increased affinity for the cytosolic ribonuclease inhibitor protein, which is anionic. Thus, Coulombic forces mediate extracellular and intracellular equilibria in a dichotomous manner that both endangers cells and defends them from the potentially lethal enzymatic activity of ribonucleases.

Fluorogenic label for biomolecular imaging.

Traditional small-molecule fluorophores are always fluorescent. This attribute can obscure valuable information in biological experiments. Here, we report on a versatile "latent" fluorophore that overcomes this limitation. At the core of the latent fluorophore is a derivative of rhodamine in which one nitrogen is modified as a urea. That modification enables rhodamine to retain half of its fluorescence while facilitating conjugation to a target molecule. The other nitrogen of rhodamine is modified with a "trimethyl lock", which enables fluorescence to be unmasked fully by a single user-designated chemical reaction. An esterase-reactive latent fluorophore was synthesized in high yield and attached covalently to a cationic protein. The resulting conjugate was not fluorescent in the absence of esterases. The enzymatic activity of esterases in endocytic vesicles and the cytosol educed fluorescence, enabling the time-lapse imaging of endocytosis into live human cells and thus providing unprecedented spatiotemporal resolution of this process. The modular design of this "fluorogenic label" enables the facile synthesis of an ensemble of small-molecule probes for the illumination of numerous biochemical and cell biological processes.

Domains within the GARP subunit Vps54 confer separate functions in complex assembly and early endosome recognition.

Tethering complexes contribute to the specificity of membrane fusion by recognizing organelle features on both donor and acceptor membranes. The Golgi-associated retrograde protein (GARP) complex is required for retrograde traffic from both early and late endosomes to the trans-Golgi network (TGN), presenting a paradox as to how a single complex can interact specifically with vesicles from multiple upstream compartments. We have found that a subunit of the GARP complex, Vps54, can be separated into N- and C-terminal regions that have different functions. Whereas the N-terminus of Vps54 is important for GARP complex assembly and stability, a conserved C-terminal domain mediates localization to an early endocytic compartment. Mutation of this C-terminal domain has no effect on retrograde transport from late endosomes. However, a specific defect in retrieval of Snc1 from early endosomes is observed when recycling from late endosomes to the Golgi is blocked. These data suggest that separate domains recruit tethering complexes to different upstream compartments to regulate individual trafficking pathways.