Microfluidic Platform for the Isolation of Cancer-Cell Subpopulations Based on Single-Cell Glycolysis.

High rates of glycolysis in tumors have been associated with cancer metastasis, tumor recurrence, and poor outcomes. In this light, single cells that exhibit high glycolysis are specific targets for therapy. However, the study of these cells requires efficient tools for their isolation. We use a droplet microfluidic technique developed in our lab, Sorting by Interfacial Tension (SIFT), to isolate cancer cell subpopulations based on glycolysis without the use of labels or active sorting components. By controlling the flow conditions on chip, the threshold of selection can be modified, enabling the isolation of cells with different levels of glycolysis. Hypoxia in tumors, that can be simulated with treatment with CoCl2, leads to an increase in glycolysis, and more dangerous tumors. The device was used to enrich CoCl2 treated MDA-MB 231 breast cancer cells from an untreated population. It is also used to sort K562 human chronic myelogenous leukemia cells that have either been treated or untreated with 2-deoxy-d-glucose (2DG), a pharmaceutical that targets cell metabolism. The technique provides a facile and robust way of separating cells based on elevated glycolytic activity; a biomarker associated with cancer cell malignancy.

Impact of early disease progression and surgical complications on adjuvant chemotherapy completion rates and survival in patients undergoing the surgery first approach for resectable pancreatic ductal adenocarcinoma - A population-based cohort study.

BACKGROUND: Multimodality treatment (MMT) improves survival for patients with pancreatic ductal adenocarcinoma (PDAC). The surgery-first (SF) strategy is the most universally accepted approach. MATERIAL AND METHODS: Population-based retrospective cohort study of all cases of resectable PDAC from 2006 to 2012. Patients were planned for adjuvant chemotherapy (AC) with the Nordic 5-fluorouracil/leucovorin regimen. Reasons for and rates of failure to complete AC, postoperative major complications (PMC), and overall survival (OS) were analysed. RESULTS: Of 203 patients, 85 (41.9%) completed AC, 41 (20.2%) failed to complete AC, and 77 (37.9%) never initiated AC. Primary reasons for not initiating or completing AC were early disease progression (34.7%), postoperative complications/poor performance status (32.2%), and age > 75 years (24.6%). Median OS in the whole cohort was 17.0 months, and 20.0 months in patients who initiated AC. Median OS in patients who completed AC was higher than in patients who did not (25.0 months vs. 12.0 months, p < 0.001). PMC (n = 41) were associated with decreased initiation rate (p < 0.001) and completion rate (p = 0.007) of AC, and decreased median OS (11.0 months vs. 19.0 months, p = 0.028). Among patients with R1 resection, PMC again were associated with worse median OS (8.0 months vs. 16.0 months, p = 0.028). Multivariate analysis demonstrated that completion of MMT and tumour grade (G1/G2) were related to mortality rate (p < 0.001). Mortality risk for patients who completed AC was reduced also when adjusting for competing risk (SHR 0.426, p < 0.001). CONCLUSIONS: MMT completion is strongly associated with reduced mortality risk in patients with resectable PDAC undergoing the SF approach. Early disease progression and PMC/poor performance status preclude MMT completion in more than one third of the patients. These reasons for failure to complete MMT underscore the need for strategies to improve patient selection and reduce surgical morbidity in patients with resectable PDAC.

Potential airborne transmission between two isolation cubicles through a shared anteroom.

Full-scale experiments and CFD simulations were performed to study potential inter-cubicle airborne transmissions through a shared anteroom due to the hinged door opening. When doors are closed, current negative pressure designs are effective for the containment of airborne pathogens in the 'dirty' cubicle with an index patient. When the 'dirty' cubicle door is open, airborne agents can move into the other 'clean' cubicle via the shared anteroom. As the door being opened or closed, the door sweeping effect is the main source of the two-way airflow and contaminant exchange through the doorway. When the dirty cubicle door remains fully open, temperature difference and concentration gradient across the doorway induce the two-way buoyancy-driven flow and transport of airborne agents across the doorway. The longer the dirty cubicle door remains fully open (10 s, 30 s or 60 s) or the smaller the air change rate (34-8.5 ACH for each cubicle), the more airborne pathogens are being transported into the 'clean' cubicle and the longer time it takes to remove them after the door is closed. Keeping the door completely open is potentially responsible for the majority of inter-cubicle transmissions if its duration is much longer than the duration of door motion (only 3 s). Our analyses suggest a potential inter-cubicle infection risk if the shared anteroom is used for multiple isolation cubicles. Decreasing the duration of door opening, raising air change rate or using a curtain at the doorway are recommended to reduce inter-cubicle exposure hazards.

Polymorphonuclear leukocytes mediate Staphylococcus aureus Panton-Valentine leukocidin-induced lung inflammation and injury.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is epidemic in the United States, even rivaling HIV/AIDS in its public health impact. The pandemic clone USA300, like other CA-MRSA strains, expresses Panton-Valentine leukocidin (PVL), a pore-forming toxin that targets polymorphonuclear leukocytes (PMNs). PVL is thought to play a key role in the pathogenesis of necrotizing pneumonia, but data from rodent infection models are inconclusive. Rodent PMNs are less susceptible than human PMNs to PVL-induced cytolysis, whereas rabbit PMNs, like those of humans, are highly susceptible to PVL-induced cytolysis. This difference in target cell susceptibility could affect results of experimental models. Therefore, we developed a rabbit model of necrotizing pneumonia to compare the virulence of a USA300 wild-type strain with that of isogenic PVL-deletion mutant and -complemented strains. PVL enhanced the capacity of USA300 to cause severe lung necrosis, pulmonary edema, alveolar hemorrhage, hemoptysis, and death, hallmark clinical features of fatal human necrotizing pneumonia. Purified PVL instilled directly into the lung caused lung inflammation and injury by recruiting and lysing PMNs, which damage the lung by releasing cytotoxic granule contents. These findings provide insights into the mechanism of PVL-induced lung injury and inflammation and demonstrate the utility of the rabbit for studying PVL-mediated pathogenesis.

Lifetime Enhancement and Degradation Study of Blue OLEDs Using Deuterated Materials.

Significant lifetime enhancement, up to an eight-fold increase in T90, has been demonstrated in blue organic light-emitting diode (OLED) devices through the deuteration of host and hole transport materials. We observed a progressive increase in T90 using a series of anthracene-based hydrocarbon hosts with incremental deuteration in the emitting layer. In addition, we realized further lifetime improvement using a deuterated hole-transport layer along with the deuterated emitting layer. To elucidate the deuteration effects, we utilized laser desorption/ionization-time-of-flight (LDI-TOF) mass spectrometry for in situ UV irradiation to induce photodegradation and immediate chemical analysis of the resultant photodegradation species. Adducts between the host and moieties from transport materials were identified in UV-degraded films comprising a mixture of host and transport materials, indicating that similar species could be produced in OLED devices using these materials. Deuteration, in effect, mediated the formation of these adduct species, presumably electroluminescence quenchers, and thus improved the device lifetime. An approximate agreement was obtained between the kinetic isotope effect of the photodegradation reactions and the enhancement in device lifetime with deuteration.

Activation of TRPC1 by STIM1 in ER-PM microdomains involves release of the channel from its scaffold caveolin-1.

Store-operated Ca(2+) entry (SOCE) is activated by redistribution of STIM1 into puncta in discrete ER-plasma membrane junctional regions where it interacts with and activates store-operated channels (SOCs). The factors involved in precise targeting of the channels and their retention at these specific microdomains are not yet defined. Here we report that caveolin-1 (Cav1) is a critical plasma membrane scaffold that retains TRPC1 within the regions where STIM1 puncta are localized following store depletion. This enables the interaction of TRPC1 with STIM1 that is required for the activation of TRPC1-SOCE. Silencing Cav1 in human submandibular gland (HSG) cells decreased plasma membrane retention of TRPC1, TRPC1-STIM1 clustering, and consequently reduced TRPC1-SOCE, without altering STIM1 puncta. Importantly, activation of TRPC1-SOCE was associated with an increase in TRPC1-STIM1 and a decrease in TRPC1-Cav1 clustering. Consistent with this, overexpression of Cav1 decreased TRPC1-STIM1 clustering and SOCE, both of which were recovered when STIM1 was expressed at higher levels relative to Cav1. Silencing STIM1 or expression of DeltaERM-STIM1 or STIM1((684)EE(685)) mutant prevented dissociation of TRPC1-Cav1 and activation of TRPC1-SOCE. However expression of TRPC1-((639)KK(640)) with STIM1((684)EE(685)) restored function and the dissociation of TRPC1 from Cav1 in response to store depletion. Further, conditions that promoted TRPC1-STIM1 clustering and TRPC1-SOCE elicited corresponding changes in SOCE-dependent NFkB activation and cell proliferation. Together these data demonstrate that Cav1 is a critical plasma membrane scaffold for inactive TRPC1. We suggest that activation of TRPC1-SOC by STIM1 mediates release of the channel from Cav1.

Characterization of obstructive sleep apnea before and after tongue-lip adhesion in children with micrognathia.

OBJECTIVES: To characterize airway obstruction before and after tongue-lip adhesion in children with micrognathia using polysomnography. DESIGN: Retrospective pilot case series. PARTICIPANTS AND METHODS: Evaluation of all children with micrognathia who underwent tongue-lip adhesion and polysomnography before and after surgery from 2002 to 2007 (N  =  8). RESULTS: Eight children met inclusion criteria; six were girls. The mean interval between polysomnography and tongue-lip adhesion was 6 days (range, 2 to 13 days) preoperatively and 17 days (range, 5 to 32 days) postoperatively. Severe obstructive sleep apnea was identified in seven of eight (88%) children, with a mean preoperative obstructive apnea hypopnea index of 52.6 events per hour (range, 7.1 to 85.7 events per hour). None had significant central sleep apneas (>5 per hour). Tongue-lip adhesion resulted in a mean decrease of 34.5 events per hour (range, -65.8 to 71.6 events per hour). After tongue-lip adhesion, seven of eight (87.5%) patients had an improved obstructive apnea hypopnea index, with resolution of obstructive sleep apnea in one child and improvement to mild (two) and moderate (two) obstructive sleep apnea in four others. Only one child had an obstructive apnea hypopnea index that increased after tongue-lip adhesion. Peak end-tidal pCO(2) measurements were elevated in all eight children before surgery at a mean of 60 mm Hg (range, 52 to 76 mm Hg) that improved to 51 mm Hg (range, 45 to 59 mm Hg), with normal peak levels in four children. Oxygen saturation nadir improved from 73% (range, 58% to 81%) to 82% (range, 65% to 94%). CONCLUSIONS: Tongue-lip adhesion may be performed in micrognathic infants to alleviate airway obstruction. Polysomnographic evaluation in this pilot study before and after surgery suggests that tongue-lip adhesion usually improves obstructive sleep apnea, but only 38% had complete resolution. Future studies of tongue-lip adhesion efficacy should include polysomnographic evaluation.

Prevalence and severity of obstructive sleep apnea and snoring in infants with Pierre Robin sequence.

OBJECTIVE: To evaluate the prevalence and severity of obstructive sleep apnea in infants with Pierre Robin sequence prior to airway intervention and determine whether snoring correlates with the presence of obstructive sleep apnea in this population. DESIGN: Retrospective case series. SETTING: Urban tertiary care teaching hospital. PARTICIPANTS/METHODS: Review of infants with Pierre Robin sequence who underwent polysomnography in the first year of life from 2002 to 2007. Only results from the initial polysomnography were analyzed. A subgroup of consecutive prospectively tested patients was also evaluated. RESULTS: A total of 33 infants with Pierre Robin sequence were identified. Of these, 13 (39%), 11 girls and two boys, underwent polysomnography in the first year of life. The mean age at evaluation was 48 days (range, 7 to 214 days). Seven nonconsecutive and six consecutive patients were included, and no significant differences were seen between groups. Obstructive sleep apnea was identified in 11 of 13 (85%) infants. The mean obstructive apnea-hypopnea index was 33.5 (range, 0 to 85.7). Obstructive sleep apnea severity was mild in 2 of 11 (18%), moderate in 3 of 11 (27%), and severe in 6 of 11 (55%). Mean end-tidal Pco(2) measurements were elevated at 59 mm Hg (range, 47 to 76 mm Hg). Mean oxygen saturation nadir was decreased at 80% (range, 68% to 93%). Snoring occurred in only 7 of 13 (54%). Of the subjects with obstructive sleep apnea, snoring occurred in 6 of 11 (55%). CONCLUSION: The high incidence of obstructive sleep apnea in this group suggests that polysomnography should be promptly performed in children with Pierre Robin sequence. Although snoring was seen in the majority, the absence of snoring did not exclude the presence of obstructive sleep apnea.

Blockchain-IoT-Driven Nursing Workforce Planning for Effective Long-Term Care Management in Nursing Homes.

Due to the global ageing population, the increasing demand for long-term care services for the elderly has directed considerable attention towards the renovation of nursing homes. Although nursing homes play an essential role within residential elderly care, professional shortages have created serious pressure on the elderly service sector. Effective workforce planning is vital for improving the efficacy and workload balance of existing nursing staff in today's complex and volatile long-term care service market. Currently, there is lack of an integrated solution to monitor care services and determine the optimal nursing staffing strategy in nursing homes. This study addresses the above challenge through the formulation of nursing staffing optimisation under the blockchain-internet of things (BIoT) environment. Embedding a blockchain into IoT establishes the long-term care platform for the elderly and care workers, thereby decentralising long-term care information in the nursing home network to achieve effective care service monitoring. Moreover, such information is further utilised to optimise nursing staffing by using a genetic algorithm. A case study of a Hong Kong nursing home was conducted to illustrate the effectiveness of the proposed system. We found that the total monthly staffing cost after using the proposed model was significantly lower than the existing practice with a change of -13.48%, which considers the use of heterogeneous workforce and temporary staff. Besides, the care monitoring and staffing flexibility are further enhanced, in which the concept of skill substitution is integrated in nursing staffing optimisation.

Natural ventilation for reducing airborne infection in hospitals.

High ventilation rate is shown to be effective for reducing cross-infection risk of airborne diseases in hospitals and isolation rooms. Natural ventilation can deliver much higher ventilation rate than mechanical ventilation in an energy-efficient manner. This paper reports a field measurement of naturally ventilated hospital wards in Hong Kong and presents a possibility of using natural ventilation for infection control in hospital wards. Our measurements showed that natural ventilation could achieve high ventilation rates especially when both the windows and the doors were open in a ward. The highest ventilation rate recorded in our study was 69.0 ACH. The airflow pattern and the airflow direction were found to be unstable in some measurements with large openings. Mechanical fans were installed in a ward window to create a negative pressure difference. Measurements showed that the negative pressure difference was negligible with large openings but the overall airflow was controlled in the expected direction. When all the openings were closed and the exhaust fans were turned on, a reasonable negative pressure was created although the air temperature was uncontrolled. The high ventilation rate provided by natural ventilation can reduce cross-infection of airborne diseases, and thus it is recommended for consideration of use in appropriate hospital wards for infection control. Our results also demonstrated a possibility of converting an existing ward using natural ventilation to a temporary isolation room through installing mechanical exhaust fans.

Method for Passive Droplet Sorting after Photo-Tagging.

We present a method to photo-tag individual microfluidic droplets for latter selection by passive sorting. The use of a specific surfactant leads to the interfacial tension to be very sensitive to droplet pH. The photoexcitation of droplets containing a photoacid, pyranine, leads to a decrease in droplet pH. The concurrent increase in droplet interfacial tension enables the passive selection of irradiated droplets. The technique is used to select individual droplets within a droplet array as illuminated droplets remain in the wells while other droplets are eluted by the flow of the external oil. This method was used to select droplets in an array containing cells at a specific stage of apoptosis. The technique is also adaptable to continuous-flow sorting. By passing confined droplets over a microfabricated trench positioned diagonally in relation to the direction of flow, photo-tagged droplets were directed toward a different chip exit based on their lateral movement. The technique can be performed on a conventional fluorescence microscope and uncouples the observation and selection of droplets, thus enabling the selection on a large variety of signals, or based on qualitative user-defined features.

The axonal membrane protein Caspr, a homologue of neurexin IV, is a component of the septate-like paranodal junctions that assemble during myelination.

We have investigated the potential role of contactin and contactin-associated protein (Caspr) in the axonal-glial interactions of myelination. In the nervous system, contactin is expressed by neurons, oligodendrocytes, and their progenitors, but not by Schwann cells. Expression of Caspr, a homologue of Neurexin IV, is restricted to neurons. Both contactin and Caspr are uniformly expressed at high levels on the surface of unensheathed neurites and are downregulated during myelination in vitro and in vivo. Contactin is downregulated along the entire myelinated nerve fiber. In contrast, Caspr expression initially remains elevated along segments of neurites associated with nascent myelin sheaths. With further maturation, Caspr is downregulated in the internode and becomes strikingly concentrated in the paranodal regions of the axon, suggesting that it redistributes from the internode to these sites. Caspr expression is similarly restricted to the paranodes of mature myelinated axons in the peripheral and central nervous systems; it is more diffusely and persistently expressed in gray matter and on unmyelinated axons. Immunoelectron microscopy demonstrated that Caspr is localized to the septate-like junctions that form between axons and the paranodal loops of myelinating cells. Caspr is poorly extracted by nonionic detergents, suggesting that it is associated with the axon cytoskeleton at these junctions. These results indicate that contactin and Caspr function independently during myelination and that their expression is regulated by glial ensheathment. They strongly implicate Caspr as a major transmembrane component of the paranodal junctions, whose molecular composition has previously been unknown, and suggest its role in the reciprocal signaling between axons and glia.

Sorting by interfacial tension (SIFT): label-free selection of live cells based on single-cell metabolism.

Selection of live cells from a population is critical in many biological studies and biotechnologies. We present here a novel droplet microfluidic approach that allows for label-free and passive selection of live cells using the glycolytic activity of individual cells. It was observed that with the use of a specific surfactant utilized to stabilize droplet formation, the interfacial tension of droplets was very sensitive to pH. After incubation, cellular lactate release results in droplets containing a live cell to attain a lower pH than other droplets. This enables the sorting of droplets containing live cells when confined droplets flow over a microfabricated trench oriented diagonally with respect to the direction of flow. The technique is demonstrated with human U87 glioblastoma cells for the selection of only droplets containing a live cell while excluding either empty droplets or droplets containing a dead cell. This label-free sorting method, dubbed sorting by interfacial tension (SIFT) presents a new strategy to sort diverse cell types based on metabolic activity.

Sorting by interfacial tension (SIFT): Label-free enzyme sorting using droplet microfluidics.

Droplet microfluidics has the ability to greatly increase the throughput of screening and sorting of enzymes by carrying reagents in picoliter droplets flowing in inert oils. It was found with the use of a specific surfactant, the interfacial tension of droplets can be very sensitive to droplet pH. This enables the sorting of droplets of different pH when confined droplets encounter a microfabricated trench. The device can be extended to sort enzymes, as a large number of enzymatic reactions lead to the production of an acidic or basic product and a concurrent change in solution pH. The progress of an enzymatic reaction is tracked from the position of a flowing train of droplets. We demonstrate the sorting of esterase isoenzymes based on their enzymatic activity. This label-free technology, that we dub droplet sorting by interfacial tension (SIFT), requires no active components and would have applications for enzyme sorting in high-throughput applications that include enzyme screening and directed evolution of enzymes.

The functional ALDH2 polymorphism is associated with breast cancer risk: A pooled analysis from the Breast Cancer Association Consortium.

BACKGROUND: Epidemiological studies consistently indicate that alcohol consumption is an independent risk factor for female breast cancer (BC). Although the aldehyde dehydrogenase 2 (ALDH2) polymorphism (rs671: Glu>Lys) has a strong effect on acetaldehyde metabolism, the association of rs671 with BC risk and its interaction with alcohol intake have not been fully elucidated. We conducted a pooled analysis of 14 case-control studies, with individual data on Asian ancestry women participating in the Breast Cancer Association Consortium. METHODS: We included 12,595 invasive BC cases and 12,884 controls for the analysis of rs671 and BC risk, and 2,849 invasive BC cases and 3,680 controls for the analysis of the gene-environment interaction between rs671 and alcohol intake for BC risk. The pooled odds ratios (OR) with 95% confidence intervals (CI) associated with rs671 and its interaction with alcohol intake for BC risk were estimated using logistic regression models. RESULTS: The Lys/Lys genotype of rs671 was associated with increased BC risk (OR = 1.16, 95% CI 1.03-1.30, p = 0.014). According to tumor characteristics, the Lys/Lys genotype was associated with estrogen receptor (ER)-positive BC (OR = 1.19, 95% CI 1.05-1.36, p = 0.008), progesterone receptor (PR)-positive BC (OR = 1.19, 95% CI 1.03-1.36, p = 0.015), and human epidermal growth factor receptor 2 (HER2)-negative BC (OR = 1.25, 95% CI 1.05-1.48, p = 0.012). No evidence of a gene-environment interaction was observed between rs671 and alcohol intake (p = 0.537). CONCLUSION: This study suggests that the Lys/Lys genotype confers susceptibility to BC risk among women of Asian ancestry, particularly for ER-positive, PR-positive, and HER2-negative tumor types.

Axon-glia interactions and the domain organization of myelinated axons requires neurexin IV/Caspr/Paranodin.

Myelinated fibers are organized into distinct domains that are necessary for saltatory conduction. These domains include the nodes of Ranvier and the flanking paranodal regions where glial cells closely appose and form specialized septate-like junctions with axons. These junctions contain a Drosophila Neurexin IV-related protein, Caspr/Paranodin (NCP1). Mice that lack NCP1 exhibit tremor, ataxia, and significant motor paresis. In the absence of NCP1, normal paranodal junctions fail to form, and the organization of the paranodal loops is disrupted. Contactin is undetectable in the paranodes, and K(+) channels are displaced from the juxtaparanodal into the paranodal domains. Loss of NCP1 also results in a severe decrease in peripheral nerve conduction velocity. These results show a critical role for NCP1 in the delineation of specific axonal domains and the axon-glia interactions required for normal saltatory conduction.

Treatment of corticosteroid refractory optic neuritis in multiple sclerosis patients with intravenous immunoglobulin.

BACKGROUND: Patients with severe visual loss because of optic neuritis refractory to high dose corticosteroids have limited therapeutic options. The use of intravenous immunoglobulin (IVIG) has been advocated in the past, but data are scarce. In this study, we use a protocol different from those used in other studies, with different timing and dosage. METHODS: Consecutive patients with corticosteroid-refractive optic neuropathy were treated with IVIG and compared with control patients who received only corticosteroids in an open-label, non-randomized, controlled prospective study. RESULTS: Twenty-three patients received treatment with IVIG and 24 matched patients who did not receive treatment with IVIG were followed as controls. All patients had visual acuity 20/400 or worse in the affected eye. There was significant improvement in the IVIG group with 18/23 (78%) subjects reaching near normal vision (20/30 or better), compared with the control group with only 3/24 (12.5%) responding similarly. CONCLUSIONS: The use of IVIG, following corticosteroids, may be useful using the protocol described herein, with sustained pulsed dosing. A larger controlled trial is indicated to confirm these results.

1700 Victims of intimate partner violence: characteristics and clinical outcomes.

OBJECTIVE: To investigate the demographics and clinical outcomes of intimate partner violence victims presenting to an emergency department. DESIGN: Retrospective, observational study. SETTING: Emergency department of a regional hospital in Hong Kong. PATIENTS: Adults presented with intimate partner violence during years 1999 to 2004. RESULTS: We assessed 1695 victims of intimate partner violence with a mean age of 39 (range, 18-84) years, of whom 87% were female. Most of the patients were in the age-group of 31 to 40 years and the overall male-to-female ratio was 1:7. In Tin Shui Wai and Yuen Long districts, such cases appeared to be on the increase. Nearly two thirds (65%) of all the victims presented to the emergency department outside the office hours of medical social workers. Approximately 10% had been abused once before, and 40% more than twice. The head (39%), face (30%), upper limbs (37%), and lower limbs (17%) were commonly the injured parts. The majority (73%) had mild injuries; severe injuries being relatively less common. The latter included lacerations or cuts (6.6%), nasal bone fractures (0.3%), limb fractures (0.8%), and ruptured tympanic membranes (0.9%). In-patient management was undertaken for 8% of the victims, due to physical injury in 68% of these individuals and psychological trauma in the remaining 32%. The hospital admission rate dropped from 12% in 2001 to 4% in 2004. CONCLUSIONS: Variations in demographic data had a significant impact on future service planning and management of intimate partner violence. Accident and Emergency Department and Emergency Medicine Ward services together with extended social worker support could provide timely, multidisciplinary care to meet the various needs of victims and subsequently reduce hospital admissions.

Metabolic characterization of serum from mice challenged with Orientia tsutsugamushi-infected mites.

Scrub typhus is an acute zoonosis caused by the obligate intracellular Gram-negative bacterium Orientia tsutsugamushi. To better understand the host response elicited by natural infection by chigger feeding, ICR mice were infected by Leptotrombidium chiangraiensis (Lc1) chiggers, and the metabolic profiles of their serum were examined over several time points after initiation of feeding. ICR mice were infected by either naive Lc1 chiggers (i.e. not infected by O. tsutsugamushi, NLc1) or O. tsutsugamushi-infected Lc1 chiggers (OLc1). Serum was collected from both groups of mice at 6 hours and 10 days after initiation of feeding. Metabolites were extracted from the serum and analysed by ultra performance liquid chromatography-tandem mass spectrometry. The resulting ion/chromatographic features were matched to a library of chemical standards for identification and quantification. Biochemicals that differed significantly between the experimental groups were identified using Welch's two-sample t tests; p ≤ 0.05 was considered statistically significant. A number of biochemicals linked to immune function were found to be significantly altered between mice infected by the NLc1 and OLc1 chiggers, including itaconate, kynurenine and histamine. Several metabolites linked to energy production were also found to be altered in the animals. In addition lipid and carbohydrate metabolism, bile acid and phospholipid homeostasis, and nucleotide metabolism were also found to be different in these two groups of mice. Markers of stress and food intake were also significantly altered. Global untargeted metabolomic characterization revealed significant differences in the biochemical profiles of mice infected by the NLc1 versus OLc1 chiggers. These findings provide an important platform for further investigation of the host responses associated with chigger-borne O. tsutsugamushi infections.

Identity of Low-Molecular-Weight Species Formed in End-To-End Cyclization Reactions Performed in THF.

Cyclic polymers were produced by end-to-end coupling of telechelic linear polymers under dilute conditions in THF, using intramolecular atom transfer radical coupling or click chemistry. In addition to the expected shift to longer elution times on gel permeation chromatography (GPC) indicative of the formation of cyclic product, lower molecular weight species were consistently observed upon analysis of the unpurified cyclization reaction mixture. By systematically removing or altering single reaction components in the highly dilute cyclization reaction, it was found that THF itself was responsible for the low-molecular-weight material, forming oligomeric chains of poly(THF) regardless of the other reaction components. When the reactions were performed at higher concentrations and for shorter time intervals, conducive to intermolecular chain-end-joining reactions, the low-molecular-weight peaks were absent. Isolation of the material and analysis by ¹H NMR confirmed that poly(THF) was being formed in the highly dilute conditions required for cyclization by end-to-end coupling. When a series of mock cyclization reactions were performed with molar ratios of the reactants held constant, but concentrations changed, it was found that lower concentrations of reactants led to higher amounts of poly(THF) side product.