Electronic Nose Development and Preliminary Human Breath Testing for Rapid, Non-Invasive COVID-19 Detection.

We adapted an existing, spaceflight-proven, robust "electronic nose" (E-Nose) that uses an array of electrical resistivity-based nanosensors mimicking aspects of mammalian olfaction to conduct on-site, rapid screening for COVID-19 infection by measuring the pattern of sensor responses to volatile organic compounds (VOCs) in exhaled human breath. We built and tested multiple copies of a hand-held prototype E-Nose sensor system, composed of 64 chemically sensitive nanomaterial sensing elements tailored to COVID-19 VOC detection; data acquisition electronics; a smart tablet with software (App) for sensor control, data acquisition and display; and a sampling fixture to capture exhaled breath samples and deliver them to the sensor array inside the E-Nose. The sensing elements detect the combination of VOCs typical in breath at parts-per-billion (ppb) levels, with repeatability of 0.02% and reproducibility of 1.2%; the measurement electronics in the E-Nose provide measurement accuracy and signal-to-noise ratios comparable to benchtop instrumentation. Preliminary clinical testing at Stanford Medicine with 63 participants, their COVID-19-positive or COVID-19-negative status determined by concomitant RT-PCR, discriminated between these two categories of human breath with a 79% correct identification rate using "leave-one-out" training-and-analysis methods. Analyzing the E-Nose response in conjunction with body temperature and other non-invasive symptom screening using advanced machine learning methods, with a much larger database of responses from a wider swath of the population, is expected to provide more accurate on-the-spot answers. Additional clinical testing, design refinement, and a mass manufacturing approach are the main steps toward deploying this technology to rapidly screen for active infection in clinics and hospitals, public and commercial venues, or at home.

COVID-19 vaccination elicits an evolving, cross-reactive antibody response to epitopes conserved with endemic coronavirus spike proteins.

The COVID-19 pandemic has triggered the first widespread vaccination campaign against a coronavirus. Many vaccinated subjects are previously naive to SARS-CoV-2; however, almost all have previously encountered other coronaviruses (CoVs), and the role of this immunity in shaping the vaccine response remains uncharacterized. Here, we use longitudinal samples and highly multiplexed serology to identify mRNA-1273 vaccine-induced antibody responses against a range of CoV Spike epitopes, in both phylogenetically conserved and non-conserved regions. Whereas reactivity to SARS-CoV-2 epitopes shows a delayed but progressive increase following vaccination, we observe distinct kinetics for the endemic CoV homologs at conserved sites in Spike S2: these become detectable sooner and decay at later time points. Using homolog-specific antibody depletion and alanine-substitution experiments, we show that these distinct trajectories reflect an evolving cross-reactive response that can distinguish rare, polymorphic residues within these epitopes. Our results reveal mechanisms for the formation of antibodies with broad reactivity against CoVs.

COVID-19 vaccination recruits and matures cross-reactive antibodies to conserved epitopes in endemic coronavirus Spike proteins.

The COVID-19 pandemic has triggered the first widespread vaccination campaign against a coronavirus. Most vaccinated subjects are naïve to SARS-CoV-2, however almost all have previously encountered other coronaviruses (CoVs) and the role of this immunity in shaping the vaccine response remains uncharacterized. Here we use longitudinal samples and highly-multiplexed serology to identify mRNA-1273 vaccine-induced antibody responses against a range of CoV Spike epitopes and in both phylogenetically conserved and non-conserved regions. Whereas reactivity to SARS-CoV-2 epitopes showed a delayed but progressive increase following vaccination, we observed distinct kinetics for the endemic CoV homologs at two conserved sites in Spike S2: these became detectable sooner, and decayed at later timepoints. Using homolog-specific depletion and alanine-substitution experiments, we show that these distinctly-evolving specificities result from cross-reactive antibodies as they mature against rare, polymorphic residues within these epitopes. Our results reveal mechanisms for the formation of antibodies with broad reactivity against CoVs.

An automated chemiluminescent immunoassay (CLIA) detects SARS-CoV-2 neutralizing antibody levels in COVID-19 patients and vaccinees.

OBJECTIVES: A specific and sensitive automated chemiluminescent immunoassay (CLIA) was developed to detect neutralizing antibody (NAb) levels. This assay can be used for the diagnosis of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection, treatment and vaccine evaluation. METHODS: The SARS-CoV-2 receptor-binding domain (RBD) and a stabilized version of the spike ectodomain as antigens were detected by CLIA. Sera NAb titers and concentrations from 860 SARS-CoV-2 vaccinees, 232 SARS-CoV-2 convalescent patients and 675 healthy individuals were tested by microneutralization test (MNT) and CLIA, respectively. Mathematical models were established to evaluate the relationship between two variables in different groups. CONCLUSIONS: With the RBD-based CLIA protocol, CLIA can be used to replace MNT to test SARS-CoV-2 NAb. Vaccine effectiveness, protectiveness and durability can be evaluated effectively by mathematical models. It is RESULTS: Analysing the relationship between NAb titers and concentrations, R2 for the decision-making tree was 0.870 and that of progressive linear fitting was 0.821. The receiver operating characteristic curve indicated specificity of 78.1%, sensitivity of 87.4%, cut-off value of 6.43 AU/mL and borderline range of 5.79-7.07 AU/mL for CLIA. Three-quarters (75.4%) of vaccinees were found to be NAb positive, and 5.35% vaccinees had NAb protective capability. The half-life of NAb in vaccinees was 10-11 weeks.for vaccinees to take a NAb assay periodically.

A Novel Patient Case Report to Show the Successful Termination of Untreatable Androgen-independent Prostate Cancer: Treatment with Cabergoline (Dopamine agonist).

INTRODUCTION: Testosterone promotes the initial development of androgen-dependent prostate cancer. This is the basis for androgen ablation treatment, which attenuates, but does not terminate, the malignancy. Instead, it leads to prolactin-dependent malignancy; in which patient death generally occurs within 5 years. This report describes the novel treatment of a patient; which terminated androgen-independent prostate cancer. RESULTS: Patient "XY" was diagnosed with prostate malignancy and metastases. He received hormonal androgen ablation treatment, chemotherapy, and radiation treatment. He developed androgen-independent prostate cancer; with expected death in 2-3 years. He was treated with cabergoline (dopamine agonist) treatment, which decreased the plasma prolactin 88%; by inhibiting the pituitary production of prolactin. The subsequent PET scan (positron emission tomography) revealed the absence of malignancy; and the CTC (circulating tumor cells) decreased from count=5.4 to count=0. DISCUSSION: The cause of androgen-independent malignancy has been unknown, and an effective chemotherapy did not exist. The activities of normal and malignant prostate cells are regulated primarily by testosterone. When testosterone availability diminishes; prolactin regulation is manifested. This is represented when androgen ablation results in the development of prolactin-dependent malignancy. An effective chemotherapy would be targeted to eliminate the plasma prolactin-manifestation of the androgen-independent malignancy. CONCLUSIONS: This report of a novel chemotherapy for androgen-independent malignancy corroborates our understanding of the implications of prolactin in its development and treatment. There are about 165,000 cases/year with 25,000 deaths/year in the U.S.; and 1.0 million cases/year with 260,000 deaths/year worldwide. Those patients with androgen-independent prostate cancer can now employ this cabergoline treatment to prevent or terminate this deadly type of prostate cancer.

Cox2p of yeast cytochrome oxidase assembles as a stand-alone subunit with the Cox1p and Cox3p modules.

Cytochrome oxidase (COX) is a hetero-oligomeric complex of the mitochondrial inner membrane that reduces molecular oxygen to water, a reaction coupled to proton transfer from the mitochondrial matrix to the intermembrane space. In the yeast Saccharomyces cerevisiae, COX is composed of 11-13 different polypeptide subunits. Here, using pulse labeling of mitochondrial gene products in isolated yeast mitochondria, combined with purification of tagged COX subunits and ancillary factors, we studied the Cox2p assembly intermediates. Analysis of radiolabeled Cox2p obtained in pulldown assays by native gel electrophoresis revealed the existence of several assembly intermediates, the largest of which had an estimated mass of 450-550 kDa. None of the other known subunits of COX were present in these Cox2p intermediates. This was also true for the several ancillary factors having still undefined functions in COX assembly. In agreement with earlier evidence, Cox18p and Cox20p, previously shown to be involved in processing and in membrane insertion of the Cox2p precursor, were found to be associated with the two largest Cox2p intermediates. A small fraction of the Cox2p module contained Sco1p and Coa6p, which have been implicated in metalation of the binuclear copper site on this subunit. Our results indicate that following its insertion into the mitochondrial inner membrane, Cox2p assembles as a stand-alone protein with the compositionally more complex Cox1p and Cox3p modules.

Press-pulse: a novel therapeutic strategy for the metabolic management of cancer.

BACKGROUND: A shift from respiration to fermentation is a common metabolic hallmark of cancer cells. As a result, glucose and glutamine become the prime fuels for driving the dysregulated growth of tumors. The simultaneous occurrence of "Press-Pulse" disturbances was considered the mechanism responsible for reduction of organic populations during prior evolutionary epochs. Press disturbances produce chronic stress, while pulse disturbances produce acute stress on populations. It was only when both disturbances coincide that population reduction occurred. METHODS: This general concept can be applied to the management of cancer by creating chronic metabolic stresses on tumor cell energy metabolism (press disturbance) that are coupled to a series of acute metabolic stressors that restrict glucose and glutamine availability while also stimulating cancer-specific oxidative stress (pulse disturbances). The elevation of non-fermentable ketone bodies protect normal cells from energy stress while further enhancing energy stress in tumor cells that lack the metabolic flexibility to use ketones as an efficient energy source. Mitochondrial abnormalities and genetic mutations make tumor cells vulnerable metabolic stress. RESULTS: The press-pulse therapeutic strategy for cancer management is illustrated with calorie restricted ketogenic diets (KD-R) used together with drugs and procedures that create both chronic and intermittent acute stress on tumor cell energy metabolism, while protecting and enhancing the energy metabolism of normal cells. CONCLUSIONS: Optimization of dosing, timing, and scheduling of the press-pulse therapeutic strategy will facilitate the eradication of tumor cells with minimal patient toxicity. This therapeutic strategy can be used as a framework for the design of clinical trials for the non-toxic management of most cancers.

Curcumin-Protected PC12 Cells Against Glutamate-Induced Oxidative Toxicity.

Glutamate is a major excitatory neurotransmitter present in the central nervous system. The glutamate/cystine antiporter system x c- connects the antioxidant defense with neurotransmission and behaviour. Overactivation of ionotropic glutamate receptors induces neuronal death, a pathway called excitotoxicity. Glutamate-induced oxidative stress is a major contributor to neurodegenerative diseases including cerebral ischemia, Alzheimer's and Huntington's disease. Curcuma has a wide spectrum of biological activities regarding neuroprotection and neurocognition. By reducing the oxidative damage, curcumin attenuates a spinal cord ischemia-reperfusion injury, seizures and hippocampal neuronal loss. The rat pheochromocytoma (PC12) cell line exhibits many characteristics useful for the study of the neuroprotection and neurocognition. This investigation was carried out to determine whether the neuroprotective effects of curcumin can be observed via the glutamate-PC12 cell model. Results indicate that glutamate (20 mM) upregulated glutathione peroxidase 1, glutathione disulphide, Ca2+ influx, nitric oxide production, cytochrome c release, Bax/Bcl-2 ratio, caspase-3 activity, lactate dehydrogenase release, reactive oxygen species, H 2 O 2 , and malondialdehyde; and downregulated glutathione, glutathione reductase, superoxide dismutase and catalase, resulting in enhanced cell apoptosis. Curcumin alleviates all these adverse effects. Conclusively, curcumin can effectively protect PC12 cells against the glutamate-induced oxidative toxicity. Its mode of action involves two pathways: the glutathione-dependent nitric oxide-reactive oxygen species pathway and the mitochondria-dependent nitric oxide-reactive oxygen species pathway.

Circulating levels of gonadotropins before and after prostate ablation in cancer patients.

UNLABELLED: Abstract Background: The prostate gland synthesizes a host of hormones, prostaglandins and growth factors. It is not clear if such biochemical factors modulate the hypothalamic/pituitary (H-P) gonadal axis. AIM: To determine the influence of the prostate gland on the hypothalamic-pituitary axis feedback system and changes in circulating hormone levels before and after cryoablation of the entire prostate. METHODS: In 37 consecutive patients with localized prostate cancer who underwent total cryoablation we investigated the circulating levels of testosterone, estradiol, progesterone, sex hormone binding globulin (SHBG) luteinizing hormone (LH), follicle stimulating hormone (FSH), dehydroepiandrosterone sulfate (DHEAS), prolactin, insulin-like growth factor-1(IGF-1) and prostatic specific antigen (PSA) from 3 years prior to diagnosis to 3 years after initiating treatment. RESULTS: The levels of LH and FSH were significantly elevated in most patients up to 3 years after surgery. No significant changes were observed for the other hormones, with a decrease in PSA as expected. CONCLUSION: LH and FSH levels rise after ablation of the prostate gland and the levels remain high for up to 3 years after surgery, which suggests that the prostate gland may influence the H-P feedback axis.

Persistent organic pollutants in serum and several different fat compartments in humans.

BACKGROUND: Chemicals that store in lipid-rich compartments have the potential for long-term disruption of metabolic and endocrine processes. Given the evidence that persistent organic pollutants (POPs) also alter systemic metabolic, endocrine, and immune system functions, it follows that elevated chemical concentrations in intra-abdominal fat may alter function, through local chemical signaling, of visceral organs. Despite this potential, there has been little study defining POP concentrations in live human intra-abdominal fat. It is at present uncertain whether POPs distribute equally to all fat compartments, including fat in serum. METHODS: Seven human subjects scheduled for elective surgery for benign lesions or cancer provided consent for removal of samples of subcutaneous and intra-abdominal fat and/or cancerous tissue. These samples were analyzed for 22 chlorinated pesticides and 10 polychlorinated biphenyl (PCB) congeners by GC/ECD plus GC/MS. RESULTS: In only two subjects were the patterns and relative concentrations of PCBs and pesticides about the same in all fat compartments. In the other subjects, there were major differences in levels in subcutaneous as compared to other compartments, but with some higher and some lower. While the pattern of PCBs in the various compartments matched that of the pesticides in some, it was opposite in others. INTERPRETATION: These results demonstrate a complicated distribution of PCB congeners and pesticides in various lipid compartments. The difference may reflect various K(ow)s, different rates of metabolism, and/or different lengths of exposure. But the results suggest that contaminant levels in serum or even subcutaneous fat do not necessarily indicate concentrations and patterns in other kinds of adipose tissue.

Induced testosterone deficiency: from clinical presentation of fatigue, erectile dysfunction and muscle atrophy to insulin resistance and diabetes.

Over the past 60 years, androgen deprivation therapy has been the mainstay of treatment of metastatic prostate cancer. However, research findings suggest that androgen deprivation therapy inflicts serious adverse effects on overall health and reduces the quality of life. Among the adverse effects known to date are insulin resistance, diabetes, metabolic syndrome fatigue, erectile dysfunction, and cardiovascular disease. In this clinical perspective, we discuss the relationship between induced androgen deficiency and a host of pathologies in the course of treatment with androgen deprivation therapy for prostate cancer patients.

Androgen deficiency and mitochondrial dysfunction: implications for fatigue, muscle dysfunction, insulin resistance, diabetes, and cardiovascular disease.

Among the major physiological functions of steroid hormones is regulation of carbohydrate, fat, and protein metabolism. Mitochondria, through oxidative phosphorylation, play a critical role in modulating a host of complex cellular metabolic pathways to produce chemical energy to meet the metabolic demand for cellular function. Thus, androgens may regulate cellular metabolism and energy production by increased mitochondrial numbers, activation of respiratory chain components, and increased transcription of mitochondrial-encoded respiratory chain genes that code for enzymes responsible for oxidative phosphorylation. Androgen deficiency is associated with increased insulin resistance, type 2 diabetes (T2DM), metabolic syndrome, obesity, and increased overall mortality. One common link among all these pathologies is mitochondrial dysfunction. Contemporary evidence exists suggesting that testosterone deficiency (TD) contributes to mitochondrial dysfunction, including structural alterations and reduced expression and activities of metabolic enzymes. Here, we postulate that TD contributes to symptoms of fatigue, insulin resistance, T2DM, cardiovascular risk, and metabolic syndrome through a common mechanism involving impairment of mitochondrial function.

Label-free voltammetric detection using individually addressable oligonucleotide microelectrode arrays.

The utility and performance of label-free, oligonucleotide probes for reagentless detection of dilute target analytes was examined using a voltammetric transduction principle in an array format. Multistep, solid-state fabrication yielded preproduction arrays of 16 individually addressable, 30 µm diameter microelectrodes in a 30 mm × 6.5 mm × 0.5 mm dipstick disposable device. The specificity of 16 nucleotide (nt) 2'-O-methylribonucleic acid and 22 nt DNA backbone probes bound through Mg(2+)-phosphonate bridges to polypyrrole films on the microelectrodes were studied using microbial target RNAs of various lengths. Probe-specific interactions with Escherichia coli O157 H7 23S rRNA (2907 nt) and Candida albicans 18S rRNA (1788 nt) were detected at 65 and 58 fmol/mL, respectively, in volumes as low as 0.5 mL. Specificity studies showed that, for a given probe, "nontarget" transcripts can contribute to changes in the voltammetric detection signal, though with responses that never exceed 70% of the detection signal acquired for specifically designed complementary targets. These results statistically validate the use of the voltammetric microelectrode array for obtaining a "yes-no" answer on complementary specific binding. The study also identifies challenges and pitfalls for the selection strategies of oligonucleotide probes.

Proximity effect in quartz crystal microbalance.

When an object approaches a vibrating quartz crystal microbalance (QCM) the resonant frequency changes. This "proximity effect" was seen at the distance of 10 mm in air and became more pronounced as the distance decreased. This effect depends on the quality factor (Q-factor) value of a QCM, conductivity of the object, and electrical connection of the object to QCM electrodes. A special setup was constructed to test the impact of the proximity effect on a QCM. Damping fluid was placed on one side of QCM, to change the Q-factor. A conducting metal disk was brought close to the other side of the QCM exposed to air. By varying the distance between the QCM and an object (metal disk), a shift in frequency was observed. This proximity effect was largest (>200 Hz for 10 MHz QCM) when the Q-factor was low and a conducting metal disk (e.g., Cu) was electrically shorted to the proximal (nearest) QCM electrode. The finite element modeling showed that the proximity effect was likely due to interaction of the object with the fringing electromagnetic field of the QCM. A simple modified Butterworth Van-Dyke model was used to describe this effect. It must be recognized that this effect may lead to large experimental artifacts in a variety of analytical QCM applications where the Q-factor changes. Therefore, in order to avoid artifacts, QCM and similar mass acoustic devices should not operate in the low Q-factor (<1000) regime.

Development of a magnetic quartz crystal microbalance.

A new technique for measurement of magnetic properties of materials is demonstrated. It can be used for the measurement of thin magnetic films during their chemical modification. The resonance frequency of a quartz crystal microbalance (QCM) with conducting polymer (polyaniline) suspension in poly(ethylene glycol) was observed to increase with increasing the externally applied uniform dc magnetic field. Slowly sweeping the magnetic field between 0 and 3.1 T results in a frequency-field response curve. Chemical doping was done by exposing the polyaniline-emeraldine base film to HCl vapor. The change in population of free spins is reflected in increased frequency-field curve magnitude after HCl doping. Two working hypotheses explaining this observation are offered to explain how frequency of QCM with deposited magnetic film shifts with increasing intensity of the magnetic field.