A Point-of-Care Device for Fully Automated, Fast and Sensitive Protein Quantification via qPCR.

This paper presents a fully automated point-of-care device for protein quantification using short-DNA aptamers, where no manual sample preparation is needed. The device is based on our novel aptamer-based methodology combined with real-time polymerase chain reaction (qPCR), which we employ for very sensitive protein quantification. DNA amplification through qPCR, sensing and real-time data processing are seamlessly integrated into a point-of-care device equipped with a disposable cartridge for automated sample preparation. The system's modular nature allows for easy assembly, adjustment and expansion towards a variety of biomarkers for applications in disease diagnostics and personalised medicine. Alongside the device description, we also present a new algorithm, which we named PeakFluo, to perform automated and real-time quantification of proteins. PeakFluo achieves better linearity than proprietary software from a commercially available qPCR machine, and it allows for early detection of the amplification signal. Additionally, we propose an alternative way to use the proposed device beyond the quantitative reading, which can provide clinically relevant advice. We demonstrate how a convolutional neural network algorithm trained on qPCR images can classify samples into high/low concentration classes. This method can help classify obese patients from their leptin values to optimise weight loss therapies in clinical settings.

Aptasensor for Quantification of Leptin Through PCR Amplification of Short DNA-Aptamers.

Protein quantification is traditionally performed through enzyme-linked immunosorbent assay (ELISA), which involves long preparation times. To overcome this, new approaches use aptamers as an alternative to antibodies. In this paper, we present a new approach to quantify proteins with short DNA aptamers through polymerase chain reaction (PCR) resulting in shorter protocol times with comparatively improved limits of detection. The proposed method includes a novel way to quantify both the target protein and the corresponding short DNA-aptamers simultaneously, which also allows us to fully characterize the performance of aptasensors. Human leptin is used as a target protein to validate this technique, because it is considered an important biomarker for obesity-related studies. In our experiments, we achieved the lowest limit of detection of 100 pg/mL within less than 2 h, a limit affected by the dissociation constant of the leptin aptamer, which could be improved by selecting a more specific aptamer. Because of the simple and inexpensive approach, this technique can be employed for Lab-On-Chip implementations and for rapid "on-site" quantification of proteins.

Assessing a novel, lab-free, point-of-care test for SARS-CoV-2 (CovidNudge): a diagnostic accuracy study.

BACKGROUND: Access to rapid diagnosis is key to the control and management of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Laboratory RT-PCR testing is the current standard of care but usually requires a centralised laboratory and significant infrastructure. We describe our diagnostic accuracy assessment of a novel, rapid point-of-care real time RT-PCR CovidNudge test, which requires no laboratory handling or sample pre-processing. METHODS: Between April and May, 2020, we obtained two nasopharyngeal swab samples from individuals in three hospitals in London and Oxford (UK). Samples were collected from three groups: self-referred health-care workers with suspected COVID-19; patients attending emergency departments with suspected COVID-19; and hospital inpatient admissions with or without suspected COVID-19. For the CovidNudge test, nasopharyngeal swabs were inserted directly into a cartridge which contains all reagents and components required for RT-PCR reactions, including multiple technical replicates of seven SARS-CoV-2 gene targets (rdrp1, rdrp2, e-gene, n-gene, n1, n2 and n3) and human ribonuclease P (RNaseP) as sample adequacy control. Swab samples were tested in parallel using the CovidNudge platform, and with standard laboratory RT-PCR using swabs in viral transport medium for processing in a central laboratory. The primary analysis was to compare the sensitivity and specificity of the point-of-care CovidNudge test with laboratory-based testing. FINDINGS: We obtained 386 paired samples: 280 (73%) from self-referred health-care workers, 15 (4%) from patients in the emergency department, and 91 (23%) hospital inpatient admissions. Of the 386 paired samples, 67 tested positive on the CovidNudge point-of-care platform and 71 with standard laboratory RT-PCR. The overall sensitivity of the point-of-care test compared with laboratory-based testing was 94% (95% CI 86-98) with an overall specificity of 100% (99-100). The sensitivity of the test varied by group (self-referred healthcare workers 94% [95% CI 85-98]; patients in the emergency department 100% [48-100]; and hospital inpatient admissions 100% [29-100]). Specificity was consistent between groups (self-referred health-care workers 100% [95% CI 98-100]; patients in the emergency department 100% [69-100]; and hospital inpatient admissions 100% [96-100]). Point of care testing performance was similar during a period of high background prevalence of laboratory positive tests (25% [95% 20-31] in April, 2020) and low prevalence (3% [95% 1-9] in inpatient screening). Amplification of viral nucleocapsid (n1, n2, and n3) and envelope protein gene (e-gene) were most sensitive for detection of spiked SARS-CoV-2 RNA. INTERPRETATION: The CovidNudge platform was a sensitive, specific, and rapid point of care test for the presence of SARS-CoV-2 without laboratory handling or sample pre-processing. The device, which has been implemented in UK hospitals since May, 2020, could enable rapid decisions for clinical care and testing programmes. FUNDING: National Institute of Health Research (NIHR) Imperial Biomedical Research Centre, NIHR Health Protection Research Unit in Healthcare Associated Infections and Antimicrobial Resistance at Oxford University in partnership with Public Health England, NIHR Biomedical Research Centre Oxford, and DnaNudge.

Atlas of Circadian Metabolism Reveals System-wide Coordination and Communication between Clocks.

Metabolic diseases are often characterized by circadian misalignment in different tissues, yet how altered coordination and communication among tissue clocks relate to specific pathogenic mechanisms remains largely unknown. Applying an integrated systems biology approach, we performed 24-hr metabolomics profiling of eight mouse tissues simultaneously. We present a temporal and spatial atlas of circadian metabolism in the context of systemic energy balance and under chronic nutrient stress (high-fat diet [HFD]). Comparative analysis reveals how the repertoires of tissue metabolism are linked and gated to specific temporal windows and how this highly specialized communication and coherence among tissue clocks is rewired by nutrient challenge. Overall, we illustrate how dynamic metabolic relationships can be reconstructed across time and space and how integration of circadian metabolomics data from multiple tissues can improve our understanding of health and disease.

Regulation of spermatogenesis by small non-coding RNAs: role of the germ granule.

The spermatogenic process relays in highly regulated gene expression mechanisms at the transcriptional and post-transcriptional levels to generate the male gamete that is needed for the perpetuation of the species. Small non-coding RNA pathways have been determined to participate in the post-transcriptional regulatory processes of germ cells. The most important sncRNA molecules that are critically involved in spermatogenesis belong to the miRNA and piRNAs pathways as illustrated by animal models where ablation of specific protein components displays male infertility. Several elements of these regulatory pathways have been found in the nuage or germ granule, a non-membranous cytoplasmatic structure that can be seen in spermatocytes and spermatids. This notion suggests that germ granules may act as organizer centers for silencing pathways in the germline. In general, miRNAs regulate spermatogenesis through targeting and down-regulation of specific transcripts to eventually promote sperm development. However, piRNAs are powerful repressors of transposon elements expression in the spermatogenic process. Here we describe the suggested functions that miRNA and piRNAs pathways execute in the regulation of spermatogenesis and include some recent studies in the field. Despite major strides on the detailed molecular mechanisms of sncRNAs in relation to spermatogenesis, there is plenty to discover on this fascinating regulatory program.

Reprogramming of the circadian clock by nutritional challenge.

Circadian rhythms and cellular metabolism are intimately linked. Here, we reveal that a high-fat diet (HFD) generates a profound reorganization of specific metabolic pathways, leading to widespread remodeling of the liver clock. Strikingly, in addition to disrupting the normal circadian cycle, HFD causes an unexpectedly large-scale genesis of de novo oscillating transcripts, resulting in reorganization of the coordinated oscillations between coherent transcripts and metabolites. The mechanisms underlying this reprogramming involve both the impairment of CLOCK:BMAL1 chromatin recruitment and a pronounced cyclic activation of surrogate pathways through the transcriptional regulator PPARγ. Finally, we demonstrate that it is specifically the nutritional challenge, and not the development of obesity, that causes the reprogramming of the clock and that the effects of the diet on the clock are reversible.

Methods for the analysis of the sperm proteome.

Proteomics is the study of the proteins of cells or tissues. Sperm proteomics aims at the identification of the proteins that compose the sperm cell and the study of their function. The recent developments in mass spectrometry (MS) have markedly increased the throughput for the identification and study of the sperm proteins. Catalogues of spermatozoal proteins in human and in model species are becoming available laying the groundwork for subsequent research, diagnostic applications, and the development of patient-specific treatments. A wide range of MS techniques is also rapidly becoming available for researchers. This chapter describes a methodological option to study the sperm cell using MS and provides a detailed protocol to identify the proteins extracted from a Percoll-purified human sperm population and separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) using LC-MS/MS.

Circadian proteins CLOCK and BMAL1 in the chromatoid body, a RNA processing granule of male germ cells.

Spermatogenesis is a complex differentiation process that involves genetic and epigenetic regulation, sophisticated hormonal control, and extensive structural changes in male germ cells. RNA nuclear and cytoplasmic bodies appear to be critical for the progress of spermatogenesis. The chromatoid body (CB) is a cytoplasmic organelle playing an important role in RNA post-transcriptional and translation regulation during the late steps of germ cell differentiation. The CB is also important for fertility determination since mutations of genes encoding its components cause infertility by spermatogenesis arrest. Targeted ablation of the Bmal1 and Clock genes, which encode central regulators of the circadian clock also result in fertility defects caused by problems other than spermatogenesis alterations. We show that the circadian proteins CLOCK and BMAL1 are localized in the CB in a stage-specific manner of germ cells. Both BMAL1 and CLOCK proteins physically interact with the ATP-dependent DEAD-box RNA helicase MVH (mouse VASA homolog), a hallmark component of the CB. BMAL1 is differentially expressed during the spermatogenic cycle of seminiferous tubules, and Bmal1 and Clock deficient mice display significant CB morphological alterations due to BMAL1 ablation or low expression. These findings suggest that both BMAL1 and CLOCK contribute to CB assembly and physiology, raising questions on the role of the circadian clock in reproduction and on the molecular function that CLOCK and BMAL1 could potentially have in the CB assembly and physiology.

Proteomic characterization of the human sperm nucleus.

Generating a catalogue of sperm nuclear proteins is an important first step towards the clarification of the function of the paternal chromatin transmitted to the oocyte upon fertilization. With this goal, sperm nuclei were obtained through CTAB treatment and isolated to over 99.9% purity without any tail fragments, acrosome or mitochondria as assessed by optical microscopy and transmission electron microscopy. The nuclear proteins were extracted and separated in 2-D and 1-D gels and the 2-D spots and 1-D bands were excised and analysed to identify the proteins through LC-MS/MS. With this approach, 403 different proteins have been identified from the isolated sperm nuclei. The most abundant family of proteins identified are the histones, for which several novel members had not been reported previously as present in the spermatogenic cell line or in the human mature spermatozoa. More than half (52.6%) of the proteins had not been detected in the previous human whole sperm cell proteome reports. Of relevance, several chromatin-related proteins, such as zinc fingers and transcription factors, so far not known to be associated with the sperm chromatin, have also been detected. This provides additional information about the nuclear proteins that are potentially relevant for epigenetic marking, proper fertilization and embryo development.

Protamine 2 precursors and processing.

Protamine 2 (P2) is synthesised as a precursor protein (pre-P2) which by proteolysis is processed to generate the mature components of the protamine 2 family of proteins (HP2, HP3 and HP4). In infertile patients, abnormal processing of the protamine 2 precursors has been suggested by the detection of an increased presence of precursor forms. However, the presence of small detectable amounts of precursor proteins has been demonstrated also in normal sperm samples, although the variation of pre-P2 in individual human sperm cells had not yet been explored. In the present manuscript we perform a mini-review describing what is known about protamine 2 precursors and P2 processing. In addition, we by immunofluorescence demonstrate the existence of a marked variation in the presence and abundance of pre-P2 in individual sperm cells.

Protamine/DNA ratios and DNA damage in native and density gradient centrifuged sperm from infertile patients.

Protamines are the major nuclear proteins condensing DNA in the sperm nucleus. One of their proposed functions is the protection of the genetic message delivered by the sperm. To date, evidence of their involvement in DNA protection has been obtained by correlating the protamine P1/P2 ratio, protamine concentrations, or chromomycin A3 staining with DNA fragmentation. However, a correlation of the absolute protamine/DNA content with the DNA fragmentation in sperm from the same infertile patients as assessed with the comet assay has not been studied. Protamine/DNA ratios were calculated after protamine and DNA extraction, electrophoresis, and gel quantification of the protamines and DNA quantification in the sperm samples of 66 infertile patients before (native sample) and after a 2-step discontinuous PureSperm density gradient centrifuged (DGC) selection of the sperm. DNA fragmentation was assessed using the alkaline comet assay. In DGC sperm, the total protamine/DNA, P1/DNA, and P2/DNA ratios all correlated inversely with DNA damage in sperm from infertile patients. The detection of this inverse correlation between protamine/DNA ratios and DNA damage in DGC sperm adds support to the hypothesis that defective protamination is related to DNA damage in the clinically relevant subpopulation of sperm from infertile men.

Methodological advances in sperm proteomics.

Proteomics is the study of the proteins of cells or tissues. Sperm proteomics aims to identify the proteins that compose the sperm cell and the study of their function. Recent developments in mass spectrometry (MS) have markedly increased the throughput to identify and study sperm proteins. Catalogues of hundreds to thousands of spermatozoan proteins in human and in model species are becoming available setting up the basis for subsequent research, diagnostic applications and the development of specific treatments. A wide range of MS techniques are also rapidly becoming available for researchers. The present review summarises the different methodological options to study the sperm cell using MS and to provide a summary of some of the ongoing proteomic studies.

Sperm cell proteomics.

The spermatozoon is an accessible cell which can be easily purified and therefore it is particularly well suited for proteomic analysis. It is also an extremely differentiated cell with very marked genetic, cellular, functional and chromatin changes as compared to other cells, and has profound implications for fertility, embryo development and heredity. The recent developments in MS have boosted the potential for identification and study of the sperm proteins. Catalogues of hundreds to thousands of spermatozoan proteins in human and in model species are becoming available setting up the basis for subsequent research, diagnostic applications and the development of specific treatments. The present article reviews the available scientific publications dealing with the composition and function of the sperm cell using an MS proteomic approach.

Protamine 2 precursors (Pre-P2), protamine 1 to protamine 2 ratio (P1/P2), and assisted reproduction outcome.

OBJECTIVE: To determine whether the presence of protamine 2 precursors (pre-P2/P2 ratio) and the protamine 1 to protamine 2 ratio (P1/P2) are related to the assisted reproduction outcome. DESIGN: Prospective study. SETTING: Assisted Reproduction Unit and University laboratory. PATIENT(S): One hundred two infertile patients undergoing treatment at the Assisted Reproduction Unit of the Hospital Clinic of Barcelona. INTERVENTION(S): Intracytoplasmic sperm injection (ICSI) and/or IVF treatment of the infertile patients, sperm protamine analysis through electrophoresis and densitometry, and pre-P2 analysis through Western blot. MAIN OUTCOME MEASURE(S): The presence of protamine 2 precursors (pre-P2/P2 ratio), sperm P1/P2 ratio, fertilization rates by IVF and/or ICSI, and pregnancy outcome. RESULT(S): Pre-P2/P2 and P1/P2 ratios are positively associated with the pregnancy rate. In addition, the P1/P2 ratio is positively associated with the proportion of embryos obtained by IVF, but not by ICSI. The pre-P2/P2 ratio was not related to the fertilization rate. CONCLUSION(S): Decreased pre-P2/P2 and P1/P2 ratios are related to a poor pregnancy outcome, but not with the proportion of embryos obtained after ISCI.

A common protamine 1 promoter polymorphism (-190 C->A) correlates with abnormal sperm morphology and increased protamine P1/P2 ratio in infertile patients.

It is known that targeting the protamine 1 gene in mice leads to infertility, abnormal chromatin packaging, and abnormal sperm morphology. Because many infertile patients also have an abnormal sperm morphology and chromatin packaging, the human protamine 1 gene (PRM1) is an important candidate to screen for potential mutations. In this work, we have screened the PRM1 gene in search of potential mutations and determined the sperm morphology and the ratio between protamine 1 and protamine 2 (P1/P2 ratio). Direct sequencing of the PRM1 promoter led to the identification of a common single-nucleotide polymorphism (SNP; -190 C-->A). The -190 AA genotype was detected at a higher frequency (13.8%) in patients with markedly altered sperm morphology (A change was also consistently higher (.331) in infertile patients with a markedly altered morphology compared with population controls (.178; P < .01). Additionally, we have determined that the P1/P2 ratio is significantly increased in patients with the PRM1 -190 AA genotype compared with patients with the CA or CC genotypes (P = .006, Mann-Whitney). These findings indicate that the common PRM1 -190 C-->A polymorphism identified is associated with abnormal sperm head morphology and abnormal P1/P2 ratio in infertile patients.

Identification of proteomic differences in asthenozoospermic sperm samples.

BACKGROUND: Asthenozoospermia is one of the most common findings present in infertile males, but its aetiology remains unknown in most cases. Present proteomic tools now offer the opportunity to identify proteins which are differentially expressed in asthenozoospermic semen samples and potentially involved in infertility. METHODS: We compared the expression of 101 sperm protein spots in 20 asthenozoospermic samples to that of 10 semen donor controls using two-dimensional proteomic analysis. RESULTS: Seventeen protein spots have been identified at different amounts in the asthenozoospermic samples compared with controls. These are cytoskeletal actin-B, annexin-A5, cytochrome C oxidase-6B, histone H2A, prolactin-inducible protein and precursor, calcium binding protein-S100A9 (2 spots), clusterin precursor, dihydrolipoamide dehydrogenase precursor, fumarate hydratase precursor, heat shock protein-HSPA2, inositol-1 monophosphatase, 3-mercapto-pyruvate sulfurtransferase/dienoyl-CoA isomerase precursor, proteasome subunit-PSMB3, semenogelin 1 precursor and testis expressed sequence 12. The detected amount of these proteins enabled the grouping of asthenozoospermic sperm samples in an unsupervised clustering analysis. CONCLUSIONS: We have identified several proteins present at different amount in asthenozoospermic sperm samples. These proteins could be candidates towards the development of diagnostic markers, and open up the opportunity to gain further insight into the pathogenic mechanisms involved in asthenozoospermia.

Marked correlations in protein expression identified by proteomic analysis of human spermatozoa.

The present work was started to explore whether a correlation could be detected among proteomic expression, protamine content and DNA integrity in human sperm cells. Towards this goal, we extracted the proteins present in the sperm cells from 47 sperm samples from infertile patients and from ten semen donors, analysed each sample by 2-D gel electrophoresis, and quantified the expression of 101 spots identified by MALDI-TOF analysis. Additionally, the protamine content and DNA integrity were also determined. Several interesting proteins such as transcription factors, prohibitin, heat shock and proteasome proteins have been identified. We have found that the expression of an important number of proteins (58 different 2-D spots) is correlated in independent sperm samples at high statistical significance (p<0.001 and r>0.5). Additionally, eight proteins have also been found to correlate with DNA integrity and seven with protamine content (p<0.05). To our knowledge, this is the first report describing the correlation between proteomics, DNA integrity and protamine content. It also sheds new light into the fundamental aspects of the human sperm and points to new potential proteins involved in male infertility.

Proteomics of human spermatozoa, protamine content and assisted reproduction outcome.

It is well known that alterations in the expression of the major sperm nuclear proteins (protamines) are related to infertility in man. In addition, other minor proteins extracted from human spermatozoa are being analysed by 2-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and identified by MALDI-TOF MS analysis. The function of the identified proteins turns out to be energy production, transcription, protein synthesis, transport, folding and turnover, cell cycle, apoptosis and oxidative stress, signal transduction, cytoskeleton, flagella and cell movement, cell recognition, metabolism and unknown function. Many of the proteins identified using MALDI-TOF had not been yet been described as being expressed in human spermatozoa. Substantial differences have been detected in the levels of some of the newly identified human sperm proteins in the different groups of infertile patients. Present research efforts are targeting the potential correlations among changes in the proteomic composition, protamine content, DNA integrity and assisted reproduction outcome.