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.

Meta-analysis of the effect of colchicine on C-reactive protein in patients with acute and chronic coronary syndromes.

OBJECTIVE: The anti-inflammatory drug colchicine has recently shown benefits in the prevention of major adverse cardiovascular events (MACE) in patients with the acute coronary syndrome (ACS) and chronic coronary syndromes (CCS). This meta-analysis focuses on understanding Colchicine's effects on the high-sensitivity C-reactive protein (hs-CRP) to provide mechanistic insight to explain its clinical event reduction. METHODS: A computerized search of MEDLINE was conducted to retrieve journal articles with studies performed on humans from 1 January 2005 to 1 January 2022, using keywords: 'Colchicine AND Coronary', 'Colchicine AND CRP', and 'Colchicine AND Coronary Artery Disease'. Studies were included if they measured hs-CRP changes from baseline, and colchicine or placebo were given to patients with ACS or CCS. RESULTS: Thirteen studies with a biomarker subgroup population of 1636 patients were included in the hs-CRP meta-analysis. Of those 13 studies, 8 studies with a total population of 6016 reported clinical events defined as myocardial infarction (MI), stroke, cardiovascular death, periprocedural MI, repeat angina after PCI and repeat revascularization. Multivariate analysis revealed a weak negative correlation of -0.1056 ( P = 0.805) between change in CRP and clinical events. Overall, colchicine treatment resulted in a greater reduction in hs-CRP levels compared with placebo (Mean Difference: -1.59; 95% Confidence Interval, -2.40 to -0.79, P = 0.0001) and clinical events (Odds Ratio: 0.78; 95% Confidence Interval 0.64 to 0.95, P = 0.01). CONCLUSION: Colchicine therapy is associated with a reduction in hs-CRP and clinical events in patients with ACS and CCS. This finding supports colchicine's anti-inflammatory efficacy via CRP reduction to explain its clinical benefit.

Estimation of Respiratory Rate from Breathing Audio.

The COVID-19 pandemic has fueled exponential growth in the adoption of remote delivery of primary, specialty, and urgent health care services. One major challenge is the lack of access to physical exam including accurate and inexpensive measurement of remote vital signs. Here we present a novel method for machine learning-based estimation of patient respiratory rate from audio. There exist non-learning methods but their accuracy is limited and work using machine learning known to us is either not directly useful or uses non-public datasets. We are aware of only one publicly available dataset which is small and which we use to evaluate our algorithm. However, to avoid the overfitting problem, we expand its effective size by proposing a new data augmentation method. Our algorithm uses the spectrogram representation and requires labels for breathing cycles, which are used to train a recurrent neural network for recognizing the cycles. Our augmentation method exploits the independence property of the most periodic frequency components of the spectrogram and permutes their order to create multiple signal representations. Our experiments show that our method almost halves the errors obtained by the existing (non-learning) methods. Clinical Relevance- We achieve a Mean Absolute Error (MAE) of 1.0 for the respiratory rate while relying only on an audio signal of a patient breathing. This signal can be collected from a smartphone such that physicians can automatically and reliably determine respiratory rate in a remote setting.

A systematic review of current trends in pharmacologic management after stent placement in nonthrombotic iliac vein lesions.

OBJECTIVE: Stenting of the iliac vein is increasingly recognized as a treatment for chronic venous insufficiency (CVI). However, the pharmacologic management after stent placement is unclear. This review was conducted to illustrate recent trends in anticoagulation and antiplatelet regimens following stent placement for nonthrombotic iliac vein lesions (NIVL). METHODS: The MEDLINE database was searched using the term "iliac vein stent." Retrieval of articles was limited to studies conducted on humans and published in English between 2010 and 2020. Studies were included that described iliac vein stent placement. Studies were excluded that contained fewer than 25 patients, performed procedures other than stent placement, did not specify the postoperative anticoagulant used, or treated lesions of thrombotic origin. RESULTS: 12 articles were included in this review, yielding a total of 2782 patients with a male-to-female ratio of 0.77. The predominant CEAP classification encountered was C3. The most common stent used in the included studies was the Wallstent (9/12), and the most common pharmacologic regimen was 3 months of clopidogrel (6/12). Warfarin, aspirin, cilostazol, and rivaroxaban were among other agents used. Primary stent patency ranged from 63.1 to 98.3%. There was no apparent correlation between pharmacologic agent used and stent patency or subjective patient outcomes. CONCLUSION: Multiple different approaches are being taken to pharmacologically manage patients following stent placement for NIVL. There is no consensus on which agent is best, nor is there a formal algorithmic approach for making this decision. Additionally, the findings in this study call into question whether anticoagulation following stenting for NIVL is necessary at all, given the similar outcomes among the different agents utilized. This review underscores the potential value of undertaking a multi-institutional prospective study to determine what is the best pharmacologic therapy following venous stent placement for NIVL.

An increase in AMPK/e-NOS signaling and attenuation of MMP-9 may contribute to remote ischemic perconditioning associated neuroprotection in rat model of focal ischemia.

Remote ischemic perconditioning (RIPerC) results in collateral enhancement and a reduction in middle cerebral artery occlusion (MCAO) induced ischemia. RIPerC likely activates multiple metabolic protective mechanisms, including effects on matrix metalloproteinases (MMPs) and protein kinases. Here we explore if RIPerC improves neuroprotection and collateral flow by modifying the activities of MMP-9 and AMPK/e-NOS. Age matched adult male Sprague Dawley rats were subjected to MCAO followed one hour later by RIPerC (3 cycles of 15 min ischemia). Animals were euthanized 24 h post-MCAO. Haematoxylin and Eosin (H&E) staining 24 h post-MCAO revealed a significant (p < 0.02) reduction in the infarction volume in RIPerC treated animals (24.9 ± 5.4%) relative to MCAO controls (42.5 ± 4.2, %). TUNEL staining showed a 42.6% reduction in the apoptotic cells with RIPerC treatment (p < 0.01). Immunoblotting in congruence with RT-PCR and Zymography showed that RIPerC significantly reduced MMP-9 expression and activity in RIPerC + MCAO group compared to MCAO group (218.3 ± 19.1% vs. 148.9 ± 12.05% (p < 0.01). Immunoblotting revealed that RIPerC was associated with a significant 2.5-fold increase in activation of p-AMPK compared to the MCAO group (p < 0.01) which was also associated with a significant increase in the e-NOS activity (p < 0.01). RIPerC resulted in reduction of infarction volume, decreased apoptotic cell death and attenuated MMP-9 activity. This together with the increased activity of p-AMPK and increase in p-eNOS may, in part explain the neuroprotection and sustained increase in blood flow observed with RIPerC following acute stroke.

Risk of Recurrence and 10-Year Outcomes in Surgically Treated Nonmelanoma Skin Cancer in Cardiac and Liver Transplant Recipients.

OBJECTIVES: To report long-term outcomes of nonmelanoma skin cancer (NMSC) in immunosuppressed cardiac and liver transplant recipients (CLTR). MATERIALS AND METHODS: The authors reviewed CLTR at the Mayo Clinic in Arizona from 1986 to 2013. Patient and tumor characteristics were recorded. Survival rates were calculated using the Kaplan-Meier method. Patient-specific and lesion-specific analyses were performed. Univariate and multivariate cox regressions were performed for comparisons. RESULTS: Seven-hundred and forty-seven patients underwent cardiac (138) or liver (609) transplantation and of these, 97 patients (13%) developed 382 invasive NMSC. The median follow-up was 11 (range, 3 to 27) years for surviving patients. Primary treatment was mainly surgery alone. At 10 years, the local recurrence (LR) rate was 20% (95% confidence interval, 15%-28%), and 14% of patients had multiple LRs. At 10 years, LR rates were higher for T3/T4 tumors when compared with T1/T2 tumors (32.5% vs. 20%, P=0.05). At 10 years, overall survival was 79% (95% confidence interval, 64%-88%). On multivariate analysis, age 61 years and more demonstrated inferior overall survival (P<0.01). CONCLUSIONS: This is the first study describing the AJCC 8th edition stage-based patterns of recurrence and long-term outcomes of surgically managed NMSC in a large cohort of immunosuppressed CLTRs. T3 and T4 tumors recur more often than early stage tumors. Further study is required to identify factors related to recurrence and guide upfront treatment intensification in this high-risk population.

Complexity in differentiating the expression of truncated or matured forms of MMP-2 and MMP-9 through zymography in rat brain tissues after acute ischaemic stroke.

Matrix metalloproteinases (MMPs) play an important role in the pathogenesis of ischaemic stroke. In particular, the mature forms of MMPs 2 and 9 have similar sizes and share gelatine as a common substrate. Both MMPs are upregulated in ischaemic stroke and play detrimental roles during stroke pathogenesis. Throughout this study, we demonstrated that pro-MMP-2 and pro-MMP-9 from ischaemic rat brain tissue homogenate is detected either through immunoblotting or zymography because of the remarkable size difference between these enzymes (72 versus 95 kDa, respectively). However, the mature MMP-2 and MMP-9 cannot be discriminated through zymography because of the almost identical sizes of these forms (66 and 67 kDa, respectively). The use of gelatine zymography on ischaemic rat brain tissue homogenate revealed a 65-kDa MMP band, corresponding to the heterogeneous band of mature MMP-2 and/or MMP-9. Furthermore, we also detected mature MMPs of 65 kDa generated from both recombinant human MMP-2 and MMP-9. Using a pull down assay in rat brain tissue homogenate with gelatine-agarose beads, we showed increased activities for both the pro and mature forms of MMP-2 and MMP-9. However, we could not determine the origin of the respective mature MMPs from the heterogeneous band. Thus, in this study, we demonstrated that the identification and quantification of mature MMP-2 and MMP-9 could not be achieved using zymography alone. Therefore, the development of a reliable technique to identify and measure the respective MMPs is needed to test new stroke therapies targeting MMP-2 and MMP-9.

Hyperthermia up-regulates matrix metalloproteinases and accelerates basement membrane degradation in experimental stroke.

Increased body temperature results in severe neuronal damage during cerebral ischemia. We hypothesized that hyperthermia hastens the degradation of basement membrane protein components and causes the disruption of brain microvasculature through early up-regulation of matrix metalloproteinases (MMPs). In this study, we present data from both non-ischemic and ischemic hemispheres of stroke induced rat brain. We found that the expression of MMP-2 and -9 and degradation of laminin and collagen IV were significantly increased in the ischemic hemisphere when compared with the non-ischemic hemisphere after 24h of normothermic stroke (p<0.001). No significant increase in MMPs expression and basement membrane components degradation was observed after 4h of normothermic stroke. At 4h, hyperthermia increased the expression of MMP-2 and -9 and subsequent degradation of laminin and collagen IV at the level that was comparable to normothermic stroke after 24h and significantly higher than 4h of normothermic stroke (p<0.001). The early increase in MMPs may be an important contributing factor to the severe neuronal damage evident with hyperthermia during an ischemic stroke.

Apolipoprotein B and triacylglycerol secretion in human triacylglycerol hydrolase transgenic mice.

Apolipoprotein B (apoB)-containing lipoproteins play a critical role in whole body lipid homeostasis and the pathogenesis of atherosclerosis. The assembly of hepatic apoB-containing lipoproteins, VLDL, is governed by the availability of lipids, including triacylglycerol (TG). The majority of TG associated with VLDL is derived from the hepatic cytoplasmic lipid stores by a process involving lipolysis followed by reesterification. Microsomal triacylglycerol hydrolase (TGH) has been demonstrated to play a role in the lipolysis/reesterification process. To evaluate the potential regulatory role of TGH in hepatic VLDL assembly, we developed inducible transgenic mice expressing a human TGH minigene under the control of the mouse metallothionein promoter. Induction of human TGH by zinc resulted in liver-specific expression of the enzyme associated with 3- to 4-fold increases in lipolytic activity that could be attenuated with a TGH-specific inhibitor. Augmented TGH activity led to increased secretion of newly synthesized apoB and plasma TG levels. These results suggest that increased hepatic expression of TGH leads to a more proatherogenic plasma lipid and apoB profile.

Mutation of F417 but not of L418 or L420 in the lipid binding domain decreases the activity of triacylglycerol hydrolase.

Human triacylglycerol hydrolase (hTGH) has been shown to play a role in hepatic lipid metabolism. Triacylglycerol hydrolase (TGH) hydrolyzes insoluble carboxylic esters at lipid/water interfaces, although the mechanism by which the enzyme adsorbs to lipid droplets is unclear. Three-dimensional modeling of hTGH predicts that catalytic residues are adjacent to an alpha-helix that may mediate TGH/lipid interaction. The helix contains a putative neutral lipid binding domain consisting of the octapeptide FLDLIADV (amino acid residues 417-424) with the consensus sequence FLXLXXXn (where n is a nonpolar residue and X is any amino acid except proline) identified in several other proteins that bind or metabolize neutral lipids. Deletion of this alpha-helix abolished the lipolytic activity of hTGH. Replacement of F417 with alanine reduced activity by 40% toward both insoluble and soluble esters, whereas replacement of L418 and L420 with alanine did not. Another potential mechanism of increasing TGH affinity for lipid is via reversible acylation. Molecular modeling predicts that C390 is available for covalent acylation. However, neither chemical modification of C390 nor mutation to alanine affected activity. Our findings indicate that F417 but not L418, L420, or C390 participates in substrate hydrolysis by hTGH.

Triacylglycerol hydrolase is localized to the endoplasmic reticulum by an unusual retrieval sequence where it participates in VLDL assembly without utilizing VLDL lipids as substrates.

The majority of hepatic intracellular triacylglycerol (TG) is mobilized by lipolysis followed by reesterification to reassemble TG before incorporation into a very-low-density lipoprotein (VLDL) particle. Triacylglycerol hydrolase (TGH) is a lipase that hydrolyzes TG within hepatocytes. Immunogold electron microscopy in transfected cells revealed a disparate distribution of this enzyme within the endoplasmic reticulum (ER), with particularly intense localization in regions surrounding mitochondria. TGH is localized to the lumen of the ER by the C-terminal tetrapeptide sequence HIEL functioning as an ER retention signal. Deletion of HIEL resulted in secretion of catalytically active TGH. Mutation of HIEL to KDEL, which is the consensus ER retrieval sequence in animal cells, also resulted in ER retention and conservation of lipolytic activity. However, KDEL-TGH was not as efficient at mobilizing lipids for VLDL secretion and exhibited an altered distribution within the ER. TGH is a glycoprotein, but glycosylation is not required for catalytic activity. TGH does not hydrolyze apolipoprotein B-associated lipids. This suggests a mechanism for vectored movement of TGs onto developing VLDL in the ER as TGH may mobilize TG for VLDL assembly, but will not access this lipid once it is associated with VLDL.

Structure-function analysis of human triacylglycerol hydrolase by site-directed mutagenesis: identification of the catalytic triad and a glycosylation site.

Triacylglycerol hydrolase is a microsomal enzyme that hydrolyzes stored cytoplasmic triacylglycerol in the liver and participates in the lipolysis/re-esterification cycle during the assembly of very-low-density lipoproteins. The structure-activity relationship of the enzyme was investigated by site-directed mutagenesis and heterologous expression. Expression of human TGH in Escherichia coli yields a protein without enzymatic activity, which suggests that posttranslational processing is necessary for the catalytic activity. Expression in baculovirus-infected Sf-9 cells resulted in correct processing of the N-terminal signal sequence and yielded a catalytically active enzyme. A putative catalytic triad consisting of a nucleophilic serine (S221), glutamic acid (E354), and histidine (H468) was identified. Site-directed mutagenesis of the residues (S221A, E354A, and H468A) yielded a catalytically inactive enzyme. CD spectra of purified mutant proteins were very similar to that of the wild-type enzyme, which suggests that the mutations did not affect folding. Human TGH was glycosylated in the insect cells. Mutagenesis of the putative N-glycosylation site (N79A) yielded an active nonglycosylated enzyme. Deletion of the putative C-terminal endoplasmic reticulum retrieval signal (HIEL) did not result in secretion of the mutant protein. A model of human TGH structure suggested a lipase alpha/beta hydrolase fold with a buried active site and two disulfide bridges (C87-C116 and C274-C285).

Heterologous expression, purification, and characterization of human triacylglycerol hydrolase.

Triacylglycerol hydrolase mobilizes stored triacylglycerol some of which is used for very-low-density lipoprotein assembly in the liver. A full-length cDNA coding for a human triacylglycerol hydrolase (hTGH) was isolated from a human liver cDNA library. The cDNA has an open reading frame of 576 amino acids with a cleavable 18-amino-acid signal sequence. The deduced amino acid sequence shows that the protein belongs to the carboxylesterase family. The hTGH was highly expressed in Escherichia coli as a 6xHis-tagged fusion protein, with the tag at the N-terminus in place of the signal peptide. However, the expressed protein was insoluble and inactive. Expression was confirmed by immunoblotting and N-terminal amino acid sequencing of the purified protein. Expression of hTGH with its native signal sequence and a C-terminal 6xHis-tag in Sf9 cells using the baculovirus expression system yielded active enzyme. N-terminal amino acid sequencing of the purified expressed protein showed correct processing of the signal peptide. The enzyme also undergoes glycosylation within the endoplasmic reticulum lumen. The results suggest that hTGH expressed in insect cells is properly folded. Therefore, baculovirus expression of hTGH and facile purification of the His-tagged enzyme will allow detailed characterization of the structure/activity relationship.