A Modified-Herringbone Micromixer for Assessing Zebrafish Sperm (MAGS).

Sperm motility analysis of aquatic model species is important yet challenging due to the small sample volume, the necessity to activate with water, and the short duration of motility. To achieve standardization of sperm activation, microfluidic mixers have shown improved reproducibility over activation by hand, but challenges remain in optimizing and simplifying the use of these microdevices for greater adoption. The device described herein incorporates a novel micromixer geometry that aligns two sperm inlet streams with modified herringbone structures that split and recombine the sample at a 1:6 dilution with water to achieve rapid and consistent initiation of motility. The polydimethylsiloxane (PDMS) chip can be operated in a positive or negative pressure configuration, allowing a simple micropipettor to draw samples into the chip and rapidly stop the flow. The device was optimized to not only activate zebrafish sperm but also enables practical use with standard computer-assisted sperm analysis (CASA) systems. The micromixer geometry could be modified for other aquatic species with differing cell sizes and adopted for an open hardware approach using 3D resin printing where users could revise, fabricate, and share designs to improve standardization and reproducibility across laboratories and repositories.

A Simple Micromilled Microfluidic Impedance Cytometer with Vertical Parallel Electrodes for Cell Viability Analysis.

Microfluidic impedance cytometry has been demonstrated as an effective platform for single cell analysis, taking advantage of microfabricated features and dielectric cell sensing methods. In this study, we present a simple microfluidic device to improve the sensitivity, accuracy, and throughput of single suspension cell viability analysis using vertical sidewall electrodes fabricated by a widely accessible negative manufacturing method. A microchannel milled through a 75 µm platinum wire, which was embedded into poly-methyl-methacrylate (PMMA), created a pair of parallel vertical sidewall platinum electrodes. Jurkat cells were interrogated in a custom low-conductivity buffer (1.2 ± 0.04 mS/cm) to reduce current leakage and increase device sensitivity. Confirmed by live/dead staining and electron microscopy, a single optimum excitation frequency of 2 MHz was identified at which live and dead cells were discriminated based on the disruption in the cell membrane associated with cell death. At this frequency, live cells were found to exhibit changes in the impedance phase with no appreciable change in magnitude, while dead cells displayed the opposite behavior. Correlated with video microscopy, a computational algorithm was created that could identify cell detection events and determine cell viability status by application of a mathematical correlation method.

Development of a Single-Piece Sperm Counting Chamber (SSCC) for Aquatic Species.

Accurate determination of sperm concentration in aquatic species is important for assisted reproduction and cryopreservation, yet is challenging as current counting methods are costly or not suitable for many species. The goal of this work was to develop a simple (single-piece and single-layer photolithography) sperm counting chamber (SSCC) for aquatic species. Goldfish (Carassius auratus) and zebrafish (Danio rerio) sperm were used for evaluation in the device, which was created with soft lithography. Four designs with different geometries were evaluated for counting accuracy. Open-corner and open-midpoint designs were the most accurate with no significant differences (P > 0.05) for most of the target sperm concentrations (0.5-1.0 × 108 cells/mL). The open-corner design was not significantly different from the Makler® counting chamber intended for human sperm cells (P = 0.6) but was significantly different from a hemocytometer (P < 0.001) intended for other cell sizes. Material cost of device production was USD 16 per unit, including photolithography supplies, glass slide and coverslip, and polydimethylsiloxane. The cost can be reduced to USD 2 per unit with repeated wafer casts. This device could be further refined for resin 3-D printing and sharing via open-hardware approaches and modified to best suit species specific applications.

Microfabrication of low-cost customisable counting chambers for standardised estimation of sperm concentration.

Evaluation of sperm concentration is essential for research and procedures involving AI, cryopreservation and sperm quality assessment. Microfabrication technologies have shown tremendous potential for rapid prototyping and fabrication of devices to assist reproduction and fertility research, but such utility has not yet been made available for most reproduction laboratories. The aim of this study was to evaluate the feasibility of using microfabrication techniques to produce counting chambers for estimation of sperm concentration. Zebrafish (Danio rerio) spermatozoa were used as a model for evaluation of functionality of the chambers. These microfabricated enumeration grid chambers (MEGC) were composed of a polydimethylsiloxane (PDMS) coverslip with grid patterns (100 µm×100 µm) and a PDMS base platform to create a known volume with a 10-µm height to restrict the cells to a single layer. The results of cell counts estimated by two of three prototype MEGC devices tested were not significantly different from the control device, a commercially available Makler chamber. The material cost for a MEGC was less than US$0.10 compared with product costs of approximately US$100 for a standard haemocytometer and US$700 for a Makler counting chamber. This study demonstrates the feasibility of microfabrication in creating low-cost counting chambers to enhance standardisation and strengthen interdisciplinary collaborations.

Genetic analysis of impaired trimethylamine metabolism using whole exome sequencing.

BACKGROUND: Trimethylaminuria (TMAU) is a genetic disorder whereby people cannot convert trimethylamine (TMA) to its oxidized form (TMAO), a process that requires the liver enzyme FMO3. Loss-of-function variants in the FMO3 gene are a known cause of TMAU. In addition to the inability to metabolize TMA precursors like choline, patients often emit a characteristic odor because while TMAO is odorless, TMA has a fishy smell. The Monell Chemical Senses Center is a research institute with a program to evaluate people with odor complaints for TMAU. METHODS: Here we evaluated ten subjects by (1) odor evaluation by a trained sensory panel, (2) analysis of their urine concentration of TMA relative to TMAO before and after choline ingestion, and (3) whole exome sequencing as well as subsequent variant analysis of all ten samples to investigate the genetics of TMAU. RESULTS: While all subjects reported they often emitted a fish-like odor, none had this malodor during sensory evaluation. However, all were impaired in their ability to produce >90% TMAO/TMA in their urine and thus met the criteria for TMAU. To probe for genetic causes, the exome of each subject was sequenced, and variants were filtered by genes with a known (FMO3) or expected effect on TMA metabolism function (other oxidoreductases). We filtered the remaining variants by allele frequency and predicated functional effects. We identified one subject that had a rare loss-of-function FMO3 variant and six with more common decreased-function variants. In other oxidoreductases genes, five subjects had four novel rare single-nucleotide polymorphisms as well as one rare insertion/deletion. Novel in this context means no investigators have previously linked these variants to TMAU although they are in dbSNP. CONCLUSIONS: Thus, variants in genes other than FMO3 may cause TMAU and the genetic variants identified here serve as a starting point for future studies of impaired TMA metabolism.

Individuals reporting idiopathic malodor production: demographics and incidence of trimethylaminuria.

BACKGROUND: Individuals with the metabolic disorder trimethylaminuria may sporadically produce malodors despite good hygiene. The psychosocial impact of trimethylaminuria can be considerable. However, trimethylaminuria is difficult to diagnose without specialized tests, in part because odor production is diet-dependent, and malodors may not be present during medical examinations. Thus, the prevalence and demographics of trimethylaminuria remain unclear. METHODS: We tested 353 patients who had unexplained (idiopathic) malodor production for trimethylaminuria using a standard choline challenge. We also collected basic demographic information. RESULTS: Approximately one third of patients (118) tested positive for trimethylaminuria. Consistent with previous reports, women, particularly African American women, were significantly overrepresented among trimethylaminuria-positive patients. Of note, the same pattern was seen among trimethylaminuria-negative patients. Also consistent with previous reports, trimethylaminuria-positive women who were still menstruating tended to produce higher levels of trimethylamine within ± 7 days of menses, although this trend was statistically marginal (P = .07). CONCLUSION: If our patient sample is representative of patients with idiopathic malodor, demographic information (race and gender) may not be useful in a differential diagnosis of trimethylaminuria. However, undiagnosed cases of trimethylaminuria may be fairly common among patients with idiopathic malodor. If so, choline challenge testing should be indicated for all such patients because trimethylaminuria is responsive to dietary and other treatments. We speculate that testing also might reveal cases of trimethylaminuria among those diagnosed with certain psychologic disorders, including olfactory reference syndrome.

The effect of menthol vapor on nasal sensitivity to chemical irritation.

INTRODUCTION: Among other effects, menthol added to cigarettes may modulate sensory response to cigarette smoke either by masking "harshness" or contributing to a desirable "impact." However, harshness and impact have been imprecisely defined and assessed using subjective measures. Thus, the current experiments used an objective measure of sensitivity to chemical irritation in the nose to test the hypothesis that menthol vapor modulates sensitivity to chemical irritation in the airways. METHODS: Nasal irritation thresholds were measured for 2 model compounds (acetic acid and allyl isothiocyanate) using nasal lateralization. In this technique, participants simultaneously sniff clean air in one nostril and chemical vapor in the other and attempt to identify the stimulated nostril. People cannot lateralize based on smell alone but can do so when chemicals are strong enough to feel. In one condition, participants were pretreated by sniffing menthol vapor. In a control condition, participants were pretreated by sniffing an odorless blank (within-subjects design). RESULTS: Pretreatment with menthol vapor decreased sensitivity to nasal irritation from acetic acid (participants required higher concentrations to lateralize) but increased sensitivity to allyl isothiocyanate (lower concentrations were required). CONCLUSIONS: The current experiments provide objective evidence that menthol vapor can modulate sensitivity to chemical irritation in the upper airways in humans. Cigarette smoke is a complex mixture of chemicals and particulates, and further work will be needed to determine exactly how menthol modulates smoking sensation. A better understanding could lead to treatments tailored to help menthol smokers quit by replacing the sensation of mentholated cigarettes.

Volatile compounds characteristic of sinus-related bacteria and infected sinus mucus: analysis by solid-phase microextraction and gas chromatography-mass spectrometry.

Volatile compounds from human breath are a potential source of information for disease diagnosis. Breath may include volatile organic compounds (VOCs) originating in the nasal sinuses. If the sinuses are infected, disease-specific volatiles may enter exhaled air. Sinus infections are commonly caused by several known bacteria. We examined the volatiles characteristic of infectious bacteria in culture using solid-phase microextraction to collect and gas chromatography-mass spectrometry as well as gas chromatography with flame photometric detection to separate and analyze the resulting VOCs. Infected sinus mucus samples were also collected and their VOCs examined. Similar characteristic volatiles were seen from both cultures of individual "pure" bacteria and several mucus samples. However, the relative amounts of characteristic VOCs from individual bacteria differ greatly between cultures and sinus mucus. New compounds, not seen in culture were also seen in some mucus samples. Our results suggest an important role for growth substrate and environment. Our data further suggests that in some sinus mucus samples identification of bacteria-specific volatiles is possible and can suggest the identity of an infecting organism to physicians. Knowledge of these bacteria-related volatiles is necessary to create electronic nose-based, volatile-specific sensors for non-invasive examination for suspected sinus infection.

Human breath odors and their use in diagnosis.

Humans emit a complex array of volatile and nonvolatile molecules that are influenced by an individual's genetics, health, diet, and stress. Olfaction is the most ancient of our distal senses and may be used to evaluate food and environmental toxins as well as recognize kin and potential predators. Many body odors evolved to be olfactory messengers, which convey information between individuals. Consequently, those practicing the healing arts have used olfaction to aid in their diagnosis of disease since the dawn of medical practice. Studies using modern instrumental analyses have focused upon analysis of breath volatiles for biomarkers of internal diseases. In these studies, a subject's oral health status appears to seldom be considered. However, saliva and properly collected alveolar air samples must pass over or come in contact with the posterior dorsal surface of the tongue, a site of bacterial plaque development and source of halitosis-related volatiles. Because of our basic research into the nature of human body odors, our lab has received referrals of people with idiopathic malodor production, from either the oral cavity or body. We developed a protocol to help differentiate individuals with chronic halitosis from those with the genetic, odor-producing metabolic disorder trimethylaminuria (TMAU). In our referred population, TMAU is the largest cause of undiagnosed body odor. Many TMAU-positive individuals present with oral symptoms of dysguesia and halitosis as well as body odor. We present data regarding the presentation of our referred subjects as well as the analytical results from a small number of these subjects regarding their oral levels of halitosis-related malodorants and trimethylamine.