Cecropins are a family of antimicrobial peptides (AMPs) that are widely found in the innate immune system of Cecropia moths. Cecropins exhibit a broad spectrum of antimicrobial and anticancer activities. The structures of Cecropins are composed of 34–39 amino acids with an N-terminal amphipathic α-helix, an AGP hinge and a hydrophobic C-terminal α-helix. KR12AGPWR6 was designed based on the Cecropin-like structural feature. In addition to its antimicrobial activities, KR12AGPWR6 also possesses enhanced salt resistance, antiendotoxin and anticancer properties. Herein, we have developed a strategy to produce recombinant KR12AGPWR6 through a salt-sensitive, pH and temperature dependent intein self-cleavage system. The His6-Intein-KR12AGPWR6 was expressed by E. coli and KR12AGPWR6 was released by the self-cleavage of intein under optimized ionic strength, pH and temperature conditions. The molecular weight and structural feature of the recombinant KR12AGPWR6 was determined by MALDI-TOF mass, CD, and NMR spectroscopy. The recombinant KR12AGPWR6 exhibited similar antimicrobial activities compared to the chemically synthesized KR12AGPWR6. Our results provide a potential strategy to obtain large quantities of AMPs and this method is feasible and easy to scale up for commercial production.

There is an urgent and imminent need to develop new agents to fight against cancer. In addition to the antimicrobial and anti-inflammatory activities, many antimicrobial peptides can bind to and lyse cancer cells. P-113, a 12-amino acid clinically active histatin-rich peptide, was found to possess anti-Candida activities but showed poor anticancer activity. Herein, anticancer activities and induced immunogenic cancer cell death of phenylalanine-(Phe-P-113), β-naphthylalanine-(Nal-P-113), β-diphenylalanine-(Dip-P-113), and β-(4,4′-biphenyl)alanine-(Bip-P-113) substituted P-113 were studied. Among these peptides, Nal-P-113 demonstrated the best anticancer activity and caused cancer cells to release potent danger-associated molecular patterns (DAMPs), such as reactive oxygen species (ROS), cytochrome c, ATP, and high-mobility group box 1 (HMGB1). These results could help in developing antimicrobial peptides with better anticancer activity and induced immunogenic cell death in therapeutic applications.

The global spread of antibiotic-resistant infections has meant that there is an urgent need to develop new antimicrobial alternatives. In this study, we developed a strategy to boost and/or synergize the activity of conventional antibiotics by combination with antimicrobial peptides tagged with the bulky non-natural amino acid β-naphthylalanine (Nal) to their N- or C-terminus. A checkerboard method was used to evaluate synergistic effects of the parent peptide and the Nal-tagged peptides. Moreover, boron-dipyrro-methene labeled vancomycin was used to characterize the synergistic mechanism of action between the peptides and vancomycin on the bacterial strains. These Nal-tagged antimicrobial peptides also reduced the antibiotic-induced release of lipopolysaccharide from Gram-negative bacteria by more than 99.95%. Our results demonstrate that Nal-tagged peptides could help in developing antimicrobial peptides that not only have enhanced antibacterial activities but also increase the synergistic effects with conventional antibiotics against antibiotic-resistant bacteria.

Electronic Medical Records (EMRs) can be used to create alerts for clinicians to identify patients at risk and to provide useful information for clinical decision-making support. In this study, we proposed a novel approach for predicting Amyotrophic Lateral Sclerosis (ALS) based on comorbidities and associated indicators using EMRs. The medical histories of ALS patients were analyzed and compared with those of subjects without ALS, and the associated comorbidities were selected as features for constructing the machine learning and prediction model. We proposed a novel weighted Jaccard index (WJI) that incorporates four different machine learning techniques to construct prediction systems. Alternative prediction models were constructed based on two different levels of comorbidity: single disease codes and clustered disease codes. With an accuracy of 83.7%, sensitivity of 78.8%, specificity of 85.7%, and area under the receiver operating characteristic curve (AUC) value of 0.907 for the single disease code level, the proposed WJI outperformed the traditional Jaccard index (JI) and scoring methods. Incorporating the proposed WJI into EMRs enabled the construction of a prediction system for analyzing the risk of suffering a specific disease based on comorbidity combinatorial patterns, which could provide a fast, low-cost, and noninvasive evaluation approach for early diagnosis of a specific disease.

Biomedical electronics plays a crucial role in bridging the gap between engineering and medicine by enabling the development of miniaturized medical equipment offering efficient diagnosis as wel l as treatment for various diseases. However, despite the recent advances in wearable biomedical devices, the key power source, the battery, is often toxic and hazardous to human beings as it can result in severe gastrointestinal injuries i f swallowed accidentally. Chargeable ingestible batteries developed in this research hold the potential of accelerating the development of safer medical technologies as they can offer high biocompatibility, non-toxicity, and global environmental sustainability. Herein, we developed a hydrogel-based ingestible batte r y which employs the selective ionic diffusion resulting due to the salinity gradient. Potassiu m chloride (KCl)-absorbed agarose hydrogels were used for creating different salinity gradients. A cation-selective gellan gu m (GG) membrane allow s the selective migration of ions within the system, hence generating an open-circuit potential difference up to 177 mV. The shapes of the mold of the batte r y were designed by a 3D printer so that it can adapt to a variety of devices. The battery can be further charged via a self-charging triboelectric nanogenerator (TENG) resulting in the generation of sufficient voltage (300 mV). We applied the electricity to stimulate the bacterial solution (containingE. coli), killing or deactivating about 90% of the bacteria just 30 minutes after the treatment. Hence, this technology is very promising for fighting antibiotic-resistant bacteria in the gastrointestinal tract, such as the oral cavity, as wel l as for providing a harmless ener g y source to medical devices.

In the absence of proper immunity, such as in the case of acquired immune deficiency syndrome (AIDS) patients, Candida albicans, the most common human fungal pathogen, may cause mucosal and even life-threatening systemic infections. P-113 (AKRHHGYKRKFH), an antimicrobial peptide (AMP) derived from the human salivary protein histatin 5, shows good safety and efficacy profiles in gingivitis and human immunodeficiency virus (HIV) patients with oral candidiasis. However, little is known about how P-113 interacts with Candida albicans or its degradation by Candida-secreted proteases that contribute to the fungi's resistance. Here, we use solution nuclear magnetic resonance (NMR) methods to elucidate the molecular mechanism of interactions between P-113 and living Candida albicans cells. Furthermore, we found that proteolytic cleavage of the C-terminus prevents the entry of P-113 into cells and that increasing the hydrophobicity of the peptide can significantly increase its antifungal activity. These results could help in the design of novel antimicrobial peptides that have enhanced stability in vivo and that can have potential therapeutic applications.

Scanning electrochemical microscopy (SECM) offers a highly sensitive and convenient approach for electrochemical mapping of interleukin-6 (IL-6), an oral cancer protein biomarker. In this study, we utilized the SECM to map the distribution of IL-6 for imaging different cancer stages on the tissue sections. The SECM results indicated that IL-6 was expressed higher in cancer tissues as compared to normal tissue and hence, SECM can be employed to improve oral cancer diagnosis. Also, IL-6 analysis can be beneficial for predicting the severity of COVID-19 at an early stage to eliminate the potential risks of serious respiratory complications in affected patients.

There is an urgent and imminent need to develop new antimicrobials to fight against antibiotic-resistant bacterial and fungal strains. In this study, a checkerboard method was used to evaluate the synergistic effects of the antimicrobial peptide P-113 and its bulky non-nature amino acid substituted derivatives with vancomycin against vancomycin-resistant Enterococcus faecium, Staphylococcus aureus, and wild-type Escherichia coli. Boron-dipyrro-methene (BODIPY) labeled vancomycin was used to characterize the interactions between the peptides, vancomycin, and bacterial strains. Moreover, neutralization of antibiotic-induced releasing of lipopolysaccharide (LPS) from E. coli by the peptides was obtained. Among these peptides, Bip-P-113 demonstrated the best minimal inhibitory concentrations (MICs), antibiotics synergism, bacterial membrane permeabilization, and supernatant LPS neutralizing activities against the bacteria studied. These results could help in developing antimicrobial peptides that have synergistic activity with large size glycopeptides such as vancomycin in therapeutic applications.

Neurofilaments: light, medium, and heavy (abbreviated as NF-L, NF-M, and NF-H, respectively), which belong to Type IV intermediate filament family (IF), are neuron-specific cytoskeletal components. Neurofilaments are axonal structural components and integral components of synapses, which are important for neuronal electric signal transmissions along the axons and post-translational modification. Abnormal assembly of neurofilaments is found in several human neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), infantile spinal muscular atrophy (SMA), and hereditary sensory-motor neuropathy (HSMN). In addition, those pathological neurofilament accumulations are known in alpha-synuclein in Parkinson's disease (PD), A beta and tau in Alzheimer's disease (AD), polyglutamine in CAG trinucleotide repeat disorders, superoxide dismutase 1 (SOD1), TAR DNA-binding protein 43 (TDP43), neuronal FUS proteins, optineurin (OPTN), ubiquilin 2 (UBQLN2), and dipeptide repeat protein (DRP) in amyotrophic lateral sclerosis (ALS). When axon damage occurs in central nervous disorders, neurofilament proteins are released and delivered into cerebrospinal fluid (CSF), which are then circulated into blood. New quantitative analyses and assay techniques are well-developed for the detection of neurofilament proteins, particularly NF-L and the phosphorylated NF-H (pNF-H) in CSF and serum. This review discusses the potential of using peripheral blood NF quantities and evaluating the severity of damage in the nervous system. Intermediate filaments could be promising biomarkers for evaluating disease progression in different nervous system disorders.

Oligomerization of protein into specific quaternary structures plays important biological functions, including regulation of gene expression, enzymes activity, and cell-cell interactions. Here, we report the determination of two crystal structures of the Grimontia hollisae (formally described as Vibrio hollisae) thermostable direct hemolysin (Gh-TDH), a pore-forming toxin. The toxin crystalized in the same space group of P21212, but with two different crystal packing patterns, each revealing three consistent tetrameric oligomerization forms called Oligomer-I, -II, and -III. A central pore with comparable depth of ~50 Å but differing in shape and size was observed in all determined toxin tetrameric oligomers. A common motif of a toxin dimer was found in all determined structures, suggesting a plausible minimum functional unit within the tetrameric structure in cell membrane binding and possible hemolytic activity. Our results show that bacterial toxins may form a single or highly symmetric oligomerization state when exerting their biological functions. The dynamic nature of multiple symmetric oligomers formed upon release of the toxin may open a niche for bacteria survival in harsh living environments.

Hypothesis Release of lipopolysaccharides (LPS) from bacteria into bloodstream may cause serious unwanted stimulation of the host immune system. P-113 is a clinically active histidine-rich antimicrobial peptide. Nal-P-113, a beta-naphthylalanine-substituted P-113, is salt-resistant but has limited LPS neutralizing activity. We suspected the size and shape of the non-natural bulky amino acid may affect its LPS neutralizing activity. Herein, antimicrobial, LPS neutralizing, and antiproteolytic effects of phenylalanine- (Phe-P-113), beta-naphthylalanine- (Nal-P-113), beta-diphenylalanine- (Dip-P-113), and beta-(4,4'-biphenyl)alanine- (Bip-P-113) substituted P-113 were studied. Experiments Structure-activity relationships of P-113, Phe-P-113, Nal-P-113, Dip-P-113, and Bip-P-113 were evaluated using antimicrobial activity assays, serum proteolytic assays, peptide-induced permeabilization of large unilamellar vesicles, zeta potential measurements, dynamic light scattering measurement of LPS aggregation, and Limulus amebocyte lysate assays for measuring LPS neutralization. In vitro and in vivo LPS neutralizing activities were further confirmed by LPS-induced inflammation inhibition in an endotoxemia mouse model. Findings Bip-P-113 and Dip-P-113 had the longest and widest non-nature amino acids, respectively. Bip-P-113 enhanced salt resistance, serum proteolytic stability, peptide-induced permeabilization, zeta potential measurements, LPS aggregation, and in vitro and in vivo LPS neutralizing activities. These results could help design novel antimicrobial peptides that have enhanced stability in vivo and that can have potential therapeutic applications. (C) 2018 Elsevier Inc. All rights reserved.

Objective: Carotid hemodynamics, such as intima-media thickness and carotid flow velocity were associated with stroke events. However, the association between carotid hemodynamics and cognitive function remains not fully clear. We aimed to investigate the relationship between carotid flow velocity and cognitive function. Design and method: A total of 744 elderly (more than 60 years) subjects completed the baseline and followed ultrasound examinations and those were evaluated cognitive function. Cognitive function was evaluated by MMSE and cognitive function impairment was defined by the MMSE less than 26. The peak-systolic velocity, end-diastolic velocity were measured in the common carotid arteries. Logistic regression was used to evaluate the association between carotid flow velocities, carotid diameters and cognitive function. Results: A total of 744 elderly subjects completed all examinations during the followed period. The prevalence Cognitive function impairment (MMSE less than 26) was 13.3% (n = 99). The peak systolic velocity (PSV) and diastolic end velocity (EDV) were lower in those with cognitive function impairment (60.5 vs. 65.5 cm/sec, p-value < 0.001 for PSV and 19.7 vs. 22.1 cm/sec, p-value < 0.001 for EDV), and only peak-systolic velocity remains significant in the multivariable models. Moreover, those with lower carotid flow velocities (the lowest 10th of peak systolic velocity) had 9.69 fold risk (95% confidence intervals: 2.75–34.21) of cognitive function impairment, compared to those with the highest 10th of peak systolic velocity. The significant association remains in the multivariable model by adjusting for age, gender, education, brachial systolic BP, fasting glucose, and low density lipoprotein cholesterol. Conclusions: Low carotid flow velocity, especially peak-systolic velocity was associated with cognitive function impairment. Lower carotid flow may involve the pathogen of cognitive function impairment in the general elderly population.

P-113, which was originally derived from the human saliva protein histatin 5, is a histidine-rich antimicrobial peptide with the sequence AKRHHGYKRKFH. P-113 is currently undergoing phase II clinical trial as a pharmaceutical agent to fight against fungal infections in HIV patients with oral candidiasis. Previously, we developed a new procedure for the high-yield expression and purification of hG31P, an analogue and antagonist of human CXCL8. Moreover, we have successfully removed lipopolysaccharide (LPS, endotoxin) associated with hG31P in the expression with Escherichia coli. In this paper, we have used hG31P as a novel fusion protein for the expression and purification of P-113. The purity of the expressed P-113 is more than 95% and the yield is 4 mg P-113 per liter of E. coli cell culture in Luria-Bertani (LB) medium. The antimicrobial activity of the purified P-113 was tested. Furthermore, we used circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy to study the structural properties of P-113. Our results indicate that using hG31P as a fusion protein to obtain large quantities of P-113 is feasible and is easy to scale up for commercial production. An effective way of producing enough P-113 for future clinical studies is evident in this study.

In this study, various types of extended gate field effect transistor (EGFET) biosensors were compared and then a model based on potential coupling between a disposable extended gate (EG) capacitor and the gate-source/drain capacitor of a nanobelt field effect transistor (NBFET) was developed and optimized for biosensor applications. Several parameters, including the dielectric thicknesses and the EG gate area coverage, the ionic strength of buffer solution, and the charge density of specific binding biomolecules, were included in the model. The potential coupling efficiency between the potential induced by surface charge on the EG and the gate voltage of the FET was analyzed and verified through pH detection. Biotin-streptavidin/avidin sensing was demonstrated with the optimized EG and NBFET. In addition, real-time measurements of the detection of botulinum neural toxin (BoNT) type-A were also performed using the EG NBFET, which could detect an extremely low concentration (20 fM) of BoNT type-A. Because the nanoelectronic field effect transistor does not need to be sacrificed after detection, the optimized EG NBFET biosensor has great potential for use in bio/chemical and point-of-care applications. (C) The Author(s) 2018. Published by ECS.

The growing number of zika virus (ZIKV) infections plus a 20-fold increase in neonatal microcephaly in newborns in Brazil have raised alarms in many countries regarding the threat to pregnant women. Instances of microcephaly and central nervous system malformations continue to increase in ZIKV outbreak regions. ZIKV is a small enveloped positive-strand RNA virus belonging to the Flavivirus genus of the Flaviviridae family. High-resolution ZIKV structures recently identified by cryo-electron microscopy indicate that the overall ZIKV structure is similar to those of other flaviviruses. With its compact surface, ZIKV is more thermally stable than the dengue virus (DENV). ZIKV E proteins have a characteristic “herringbone” structure with a single glycosylation site. The ZIKV E protein, the major protein involved in receptor binding and fusion, is formed as a head-to-tail dimer on the surfaces of viral particles. The E monomer consists of three distinct domains: DI, DII, and DIII. The finger-like DII contains a fusion loop (FL) that is inserted into the host cell endosomal membrane during pH-dependent conformational changes that drive fusion. Quaternary E:E dimer epitopes located at the interaction site of prM and E dimers can be further divided into two dimer epitopes. To date, more than 50 ZIKV vaccine candidates are now in various stages of research and development. Candidate ZIKV vaccines that are currently in phase I/II clinical trials include inactivated whole viruses, recombinant measles viral vector-based vaccines, DNA and mRNA vaccines, and a mosquito salivary peptide vaccine. Stabilized forms of ZIKV E:E dimer proteins have been successfully obtained either by introducing additional inter-subunit disulfide bond(s) in DII or via the direct assembly of E:E dimer proteins by immobilization with monomeric E proteins. The VLP-based approach is another alternative method for presenting native E:E dimer antigens among the vaccine components. Several forms of ZIKV VLPs have been reported featuring the co-expression of the prM-E, prM-E-NS1, C-prM-E, and NS2B/NS3 viral genes in human cells. To minimize the effect of the cross-reactive ADE-facilitating antibodies between ZIKV and DENV, several novel mutations have been reported either in or near the FL of DII or DIII to dampen the production of cross-reactive antibodies. Future ZIKV vaccine design efforts should be focused on eliciting improved neutralizing antibodies with a reduced level of cross-reactivity to confer sterilizing immunity.

Lipopolysaccharide (LPS, endotoxin) is the major component of Gram-negative bacterial outer surface membrane. LPS released from bacteria into bloodstream during infection may cause serious unwanted stimulation of host's immune system and lead to septic shock of the patient. Recently, we have developed a strategy to increase salt resistance and LPS neutralization of short antimicrobial peptides by adding β-naphthylalanine end-tags to their termini. Herein, correlations between membrane immersion depth, orientation, and antiendotoxin activities of the antimicrobial peptides S1 and S1-Nal-Nal have been investigated via solution structure, paramagnetic resonance enhancement, and saturation transfer difference NMR studies. Unlike the parent peptide S1, S1-Nal-Nal rotated its two terminal β-naphthylalanine residues into the hydrophobic lipid A motif of LPS micelles. The LPS-induced inflammation may then be prohibited by the blocked lipid A motif.

BACKGROUND:Many studies have investigated the association between markers for peripheral inflammation and risk of dementia, but the results have been conflicting. We aimed to evaluate the association between a specific inflammation marker, C-reactive protein (CRP), and dementia in an elderly Asian community cohort. METHODS:The cohort included 1436 individuals (ages 65 and older) from a national representative sample in Taiwan. Dementia incidence was identified using International Classification of Diseases, Ninth Revision codes for vascular dementia, Alzheimer disease, and nonvascular dementia. Baseline characteristics and CRP levels were determined. A Cox proportional hazard model and Fine and Grays model were adjusted for stroke and competing risk of death to estimate the association between inflammation and development of dementia. RESULTS:During 11.04 years (median) of follow-up, 607 individuals (50.77%) died and 260 individuals (18.11%) were diagnosed with dementia. Those with high CRP had a 55% higher risk of dementia (hazard ratio 1.55; 95% confidence interval 1.21-2.00) compared with those with normal CRP. After adjusting for possible confounding cardiovascular risk factors, high CRP was independently associated with vascular dementia but not Alzheimer disease. CONCLUSIONS:In this prospective study of an elderly Asian community cohort with more than 10 years of follow-up, the baseline serum CRP level was associated with future development of vascular dementia, but not Alzheimer disease after adjusting for common cardiovascular risk factors, stroke, and competing risk of death.

Finger trembling is a feature for neurodegenerative diseases. Accessing the trembling of fingers can help diagnosis or monitoring of progression of the disease. Currently, tremogram is used in the hospital to measure the trembling of a finger. Tremogram is a single and uniaxial acceleration gauge. Using the tremogram, only the trembling direction of a finger can be measured. The direction and the finger are determined by the physician. This work studied the possibility to employ the Leap Motion and the G-sensor/gyro to access the trembling of the five fingers. Experimental results demonstrated positive answers that a G-sensor/gyro system can access not just only those information obtained from the remogram, we can derive more information.

BACKGROUND:Asians have higher frequency of intracranial arterial stenosis. The present study aimed to compare the clinical features and outcomes of ischemic stroke patients with and without middle cerebral artery (MCA) stenosis, assessed by transcranial sonography (TCS), based on the Taiwan Stroke Registry (TSR).METHODS:Patients with acute ischemic stroke or transient ischemic attack registered in the TSR, and received both carotid duplex and TCS assessment were categorized into those with stenosis (≥50%) and without (<50%) in the extracranial internal carotid artery (ICA) and MCA, respectively. Logistic regression analysis, Kaplan-Meier method and Cox proportional hazard model were applied to assess relevant variables between groups.RESULTS:Of 6003 patients, 23.3% had MCA stenosis, 10.1% ICA stenosis, and 3.9% both MCA and ICA stenosis. Patients with MCA stenosis had greater initial NIHSS, higher likelihood of stroke-in-evolution, and more severe disability than those without (all p<0.001). Patients with MCA stenosis had higher prevalence of hypertension, diabetes and hypercholesterolemia. Patients with combined MCA and extracranial ICA stenosis had even higher NIHSS, worse functional outcome, higher risk of stroke recurrence or death (hazard ratio, 2.204; 95% confidence intervals, 1.440-3.374; p<0.001) at 3 months after stroke than those without MCA stenosis.CONCLUSIONS:In conclusion, MCA stenosis was more prevalent than extracranial ICA stenosis in ischemic stroke patients in Taiwan. Patients with MCA stenosis, especially combined extracranial ICA stenosis, had more severe neurological deficit and worse outcome.

Purpose Among the medications approved for Alzheimer’s disease (AD), rivastigmine is the only one available as transdermal patch. The aim of this study was to evaluate compliance and caregivers’ preference with oral and transdermal (rivastigmine) monotherapy in patients with mild-to-moderate AD from Taiwan. Methods Real-world Evaluation of Compliance And Preference in Alzheimer’s disease treatment (RECAP) in Taiwan was a prospective, noninterventional, observational study with a 24-week (±8 weeks) observational period for each participant. Eligible patients were grouped into one of the two treatment cohorts based on the baseline AD therapy: oral (donepezil, galantamine, rivastigmine, or memantine) or transdermal (rivastigmine patch). The primary end points were caregiver preference and caregiver assessment of patients’ compliance to the current medication (oral or transdermal medication) at Week 24 (end of the study). Safety was assessed by recording any adverse events. Results A total of 301 patients (age: 77.6±7.19 years) were enrolled from nine centers in Taiwan, of whom 138 (45.8%) patients were in the transdermal monotherapy cohort. Caregivers of patients who were exposed to both forms of therapies demonstrated a higher preference for transdermal rivastigmine monotherapy than the oral monotherapy (82.4% [n=61] versus 17.6% [n=13], P<0.0001); for patients treated with only one therapy, the caregivers’ preference was significantly in favor of the treatment to which the patient was exposed (both P<0.0001). In both cohorts, patients showed good compliance, with an overall score of 8.65±1.38 on an 11-point scale. Of 301 enrolled patients, 102 (33.9%) reported at least one adverse event during the study (51 patients each in the two cohorts). Conclusion With the higher caregiver preference and a good patient compliance, the trans-dermal rivastigmine patch is a suitable treatment choice for patients with mild-to-moderate AD, especially for patients intolerant to oral therapies.