Noise robustness and metabolic load determine the principles of central dogma regulation.

The processes of gene expression are inherently stochastic, even for essential genes required for growth. How does the cell maximize fitness in light of noise? To answer this question, we build a mathematical model to explore the trade-off between metabolic load and growth robustness. The model predicts novel principles of central dogma regulation: Optimal protein expression levels for many genes are in vast overabundance. Essential genes are transcribed above a lower limit of one message per cell cycle. Gene expression is achieved by load balancing between transcription and translation. We present evidence that each of these novel regulatory principles is observed. These results reveal that robustness and metabolic load determine the global regulatory principles that govern central dogma processes, and these principles have broad implications for cellular function.

Noise robustness and metabolic load determine the principles of central dogma regulation.

The processes of gene expression are inherently stochastic, even for essential genes required for growth. How does the cell maximize fitness in light of noise? To answer this question, we build a mathematical model to explore the trade-off between metabolic load and growth robustness. The model predicts novel principles of central dogma regulation: Optimal protein expression levels for many genes are in vast overabundance. Essential genes are transcribed above a lower limit of one message per cell cycle. Gene expression is achieved by load balancing between transcription and translation. We present evidence that each of these novel regulatory principles is observed. These results reveal that robustness and metabolic load determine the global regulatory principles that govern central dogma processes, and these principles have broad implications for cellular function.

Multiparametric Ultrasound for Diagnosing Testicular Lesions: Everything You Need to Know in Daily Clinical Practice.

BACKGROUND: Ultrasonography (US) represents the gold standard imaging method for the assessment of testicular lesions (TL). The gray-scale (GSUS) and color-Doppler (CDUS) ultrasound examination allow sonographers to investigate the size, margins, echotexture, and vascular features of TLs with the aim to differentiate benign from malignant lesions. Recently, the use of contrast-enhanced US (CEUS) and sonoelastography (SE) has led to further improvements in the differential diagnosis of TL. Although GSUS and CDUS are often sufficient to suggest the benign or malignant nature of the TL, CEUS can be decisive in the differential diagnosis of unclear findings, while SE can help to strengthen the diagnosis. The contemporary combination of GSUS, CDUS, CEUS, and SE has led to a new diagnostic paradigm named multiparametric US (mp-US), which is able to provide a more detailed characterization of TLs than single techniques alone. This narrative and pictorial review aimed to describe the mp-US appearance of several TLs. METHODS: An extensive Medline search was performed to identify studies in the English language focusing on the mp-US evaluation of TLs. RESULTS: A practical mp-US "identity card" and iconographic characterization of several benign and malignant TLs is provided herein. CONCLUSIONS: The mp-US characterization of TL reported herein can be useful in daily clinical practice.

Utility of contrast enhanced ultrasound (CEUS) in penile trauma.

Penile traumatic injuries are rare with potentially severe consequences, with clinical assessment challenging due to pain and swelling. Ultrasound is the primary imaging modality for assessing the penis. It has a high spatial resolution and rapid availability and is portable. Contrast enhanced ultrasound is an advanced ultrasound technique that adds real-time dynamic vascular imaging safely, increasing diagnostic precision and confidence, allowing for better therapeutic management of traumatic penile injuries. Contrast enhanced ultrasound helps to delineate the extent of a haematoma, distinguishing this from the herniated corpus cavernosum, detecting the point of rupture of the tunica albuginea, showing vascular abnormalities, localising a dislocated penis and determining penile viability. The integrity of the urethra may be assessed with intracavitary contrast enhanced ultrasound. Additionally, contrast enhanced ultrasound allows monitoring of the response to endovascular and surgical treatment. This review aims to detail the anatomy and pathogenesis of penile trauma, to give guidance on optimising the contrast enhanced ultrasound technique and to obtain diagnostic examinations. Illustration of the spectrum of aspects of penile trauma will be complemented with a literature review.Critical relevance statement Contrast enhanced ultrasound is a safe and efficient imaging modality that allows for highly accurate diagnoses, helping achieve more successful and conservative treatments of penile trauma complications.Key points• Ultrasound is an accurate and safe imaging test for penile trauma.• CEUS adds dynamic vascular imaging in real-time increasing diagnostic confidence.• CEUS can delineate penile haematomas, locate fractures and demonstrate vessel integrity.• CEUS potentially allows for more conservative treatment of penile trauma complications.

The one-message-per-cell-cycle rule: A conserved minimum transcription level for essential genes.

The inherent stochasticity of cellular processes leads to significant cell-to-cell variation in protein abundance. Although this noise has already been characterized and modeled, its broader implications and significance remain unclear. In this paper, we revisit the noise model and identify the number of messages transcribed per cell cycle as the critical determinant of noise. In yeast, we demonstrate that this quantity predicts the non-canonical scaling of noise with protein abundance, as well as quantitatively predicting its magnitude. We then hypothesize that growth robustness requires an upper ceiling on noise for the expression of essential genes, corresponding to a lower floor on the transcription level. We show that just such a floor exists: a minimum transcription level of one message per cell cycle is conserved between three model organisms: Escherichia coli, yeast, and human. Furthermore, all three organisms transcribe the same number of messages per gene, per cell cycle. This common transcriptional program reveals that robustness to noise plays a central role in determining the expression level of a large fraction of essential genes, and that this fundamental optimal strategy is conserved from E. coli to human cells.

The one-message-per-cell-cycle rule: A conserved minimum transcription level for essential genes.

The inherent stochasticity of cellular processes leads to significant cell-to-cell variation in protein abundance. Although this noise has already been characterized and modeled, its broader implications and significance remain unclear. In this paper, we revisit the noise model and identify the number of messages transcribed per cell cycle as the critical determinant of noise. In yeast, we demonstrate that this quantity predicts the non-canonical scaling of noise with protein abundance, as well as quantitatively predicting its magnitude. We then hypothesize that growth robustness requires an upper ceiling on noise for the expression of essential genes, corresponding to a lower floor on the transcription level. We show that just such a floor exists: a minimum transcription level of one message per cell cycle is conserved between three model organisms: Escherichia coli, yeast, and human. Furthermore, all three organisms transcribe the same number of messages per gene, per cell cycle. This common transcriptional program reveals that robustness to noise plays a central role in determining the expression level of a large fraction of essential genes, and that this fundamental optimal strategy is conserved from E. coli to human cells.

The in vivo measurement of replication fork velocity and pausing by lag-time analysis.

An important step towards understanding the mechanistic basis of the central dogma is the quantitative characterization of the dynamics of nucleic-acid-bound molecular motors in the context of the living cell. To capture these dynamics, we develop lag-time analysis, a method for measuring in vivo dynamics. Using this approach, we provide quantitative locus-specific measurements of fork velocity, in units of kilobases per second, as well as replisome pause durations, some with the precision of seconds. The measured fork velocity is observed to be both locus and time dependent, even in wild-type cells. In this work, we quantitatively characterize known phenomena, detect brief, locus-specific pauses at ribosomal DNA loci in wild-type cells, and observe temporal fork velocity oscillations in three highly-divergent bacterial species.

Beta variant COVID-19 protein booster vaccine elicits durable cross-neutralization against SARS-CoV-2 variants in non-human primates.

The rapid spread of the SARS-CoV-2 Omicron subvariants, despite the implementation of booster vaccination, has raised questions about the durability of protection conferred by current vaccines. Vaccine boosters that can induce broader and more durable immune responses against SARS-CoV-2 are urgently needed. We recently reported that our Beta-containing protein-based SARS-CoV-2 spike booster vaccine candidates with AS03 adjuvant (CoV2 preS dTM-AS03) elicited robust cross-neutralizing antibody responses at early timepoints against SARS-CoV-2 variants of concern in macaques primed with mRNA or protein-based subunit vaccine candidates. Here we demonstrate that the monovalent Beta vaccine with AS03 adjuvant induces durable cross-neutralizing antibody responses against the prototype strain D614G as well as variants Delta (B.1.617.2), Omicron (BA.1 and BA.4/5) and SARS-CoV-1, that are still detectable in all macaques 6 months post-booster. We also describe the induction of consistent and robust memory B cell responses, independent of the levels measured post-primary immunization. These data suggest that a booster dose with a monovalent Beta CoV2 preS dTM-AS03 vaccine can induce robust and durable cross-neutralizing responses against a broad spectrum of variants.

Capturing fine-grained details for video-based automation of suturing skills assessment.

OBJECTIVES: Manually-collected suturing technical skill scores are strong predictors of continence recovery after robotic radical prostatectomy. Herein, we automate suturing technical skill scoring through computer vision (CV) methods as a scalable method to provide feedback. METHODS: Twenty-two surgeons completed a suturing exercise three times on the Mimic™ Flex VR simulator. Instrument kinematic data (XYZ coordinates of each instrument and pose) were captured at 30 Hz. After standardized training, three human raters manually video segmented suturing task into four sub-stitch phases (Needle handling, Needle targeting, Needle driving, Needle withdrawal) and labeled the corresponding technical skill domains (Needle positioning, Needle entry, Needle driving, and Needle withdrawal). The CV framework extracted RGB features and optical flow frames using a pre-trained AlexNet. Additional CV strategies including auxiliary supervision (using kinematic data during training only) and attention mechanisms were implemented to improve performance. RESULTS: This study included data from 15 expert surgeons (median caseload 300 [IQR 165-750]) and 7 training surgeons (0 [IQR 0-8]). In all, 226 virtual sutures were captured. Automated assessments for Needle positioning performed best with the simplest approach (1 s video; AUC 0.749). Remaining skill domains exhibited improvements with the implementation of auxiliary supervision and attention mechanisms when deployed separately (AUC 0.604-0.794). All techniques combined produced the best performance, particularly for Needle driving and Needle withdrawal (AUC 0.959 and 0.879, respectively). CONCLUSIONS: This study demonstrated the best performance of automated suturing technical skills assessment to date using advanced CV techniques. Future work will determine if a "human in the loop" is necessary to verify surgeon evaluations.

Decreased renal cortical perfusion, independent of changes in renal blood flow and sublingual microcirculatory impairment, is associated with the severity of acute kidney injury in patients with septic shock.

BACKGROUND: Reduced renal perfusion has been implicated in the development of septic AKI. However, the relative contributions of macro- and microcirculatory blood flow and the extent to which impaired perfusion is an intrinsic renal phenomenon or part of a wider systemic shock state remains unclear. METHODS: Single-centre prospective longitudinal observational study was carried out. Assessments were made at Day 0, 1, 2 and 4 after ICU admission of renal cortical perfusion in 50 patients with septic shock and ten healthy volunteers using contrast-enhanced ultrasound (CEUS). Contemporaneous measurements were made using transthoracic echocardiography of cardiac output. Renal artery blood flow was calculated using velocity time integral and vessel diameter. Assessment of the sublingual microcirculation was made using handheld video microscopy. Patients were classified based on the degree of AKI: severe = KDIGO 3 v non-severe = KDIGO 0-2. RESULTS: At study enrolment, patients with severe AKI (37/50) had prolonged CEUS mean transit time (mTT) (10.2 vs. 5.5 s, p < 0.05), and reduced wash-in rate (WiR) (409 vs. 1203 au, p < 0.05) and perfusion index (PI) (485 vs. 1758 au, p < 0.05); differences persisted throughout the entire study. Conversely, there were no differences in either cardiac index, renal blood flow or renal resistive index. Sublingual microcirculatory variables were not significantly different between groups at study enrolment or at any subsequent time point. Although lactate was higher in the severe AKI group at study enrolment, these differences did not persist, and there were no differences in either ScvO2 or ScvCO2-SaCO2 between groups. Patients with severe AKI received higher doses of noradrenaline (0.34 vs. 0.21mcg/kg/min, p < 0.05). Linear regression analysis showed no correlation between mTT and cardiac index (R-0.18) or microcirculatory flow index (R-0.16). CONCLUSION: Renal cortical hypoperfusion is a persistent feature in critically ill septic patients who develop AKI and does not appear to be caused by reductions in macrovascular renal blood flow or cardiac output. Cortical hypoperfusion appears not be associated with changes in the sublingual microcirculation, raising the possibility of a specific renal pathogenesis that may be amenable to therapeutic intervention. Trial Registration Clinical Trials.gov NCT03713307 , 19 Oct 2018.

Protein-based SARS-CoV-2 spike vaccine booster increases cross-neutralization against SARS-CoV-2 variants of concern in non-human primates.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants that partly evade neutralizing antibodies raises concerns of reduced vaccine effectiveness and increased infection. We previously demonstrated that the SARS-CoV-2 spike protein vaccine adjuvanted with AS03 (CoV2 preS dTM-AS03) elicits robust neutralizing antibody responses in naïve subjects. Here we show that, in macaques primed with mRNA or protein-based subunit vaccine candidates, one booster dose of CoV2 preS dTM-AS03 (monovalent D614 or B.1.351, or bivalent D614 + B.1.351 formulations), significantly boosts the pre-existing neutralizing antibodies against the parental strain from 177- to 370-fold. Importantly, the booster dose elicits high and persistent cross-neutralizing antibodies covering five former or current SARS-CoV-2 variants of concern (Alpha, Beta, Gamma, Delta and Omicron) and, unexpectedly, SARS-CoV-1. Interestingly, we show that the booster specifically increases the functional antibody responses as compared to the receptor binding domain (RBD)-specific responses. Our findings show that these vaccine candidates, when used as a booster, have the potential to offer cross-protection against a broad spectrum of variants. This has important implications for vaccine control of SARS-CoV-2 variants of concern and informs on the benefit of a booster with the vaccine candidates currently under evaluation in clinical trials.

An unusual cause of free air in the abdomen: emphysematous cystitis with bladder diverticulum perforation.

A 64-year-old male, with a history of chronic urinary outflow obstruction secondary to benign prostatic hyperplasia, presented with haematuria and urinary retention following spontaneous removal of his long-term catheter. The patient was septic on admission and a CT examination of the abdomen and pelvis showed an acutely inflamed urinary bladder diverticulum and extensive intra-abdominal free air. The patient was treated medically for emphysematous cystitis centred on a perforated bladder diverticulum, which was thought to be caused by the underlying infectious/inflammatory process. Alternative aetiologies for free air in the abdomen such a traumatic bladder perforation and gastrointestinal perforation were considered and excluded. The patient responded well to medical management and was discharged after an 11 day in-patient stay.

Multiparametric ultrasound imaging characteristics of multiple testicular adrenal rest tumours in congenital adrenal hyperplasia.

INTRODUCTION: Testicular adrenal rest cell tumours (TART) are rare benign adrenocorticotropic hormone-dependent testicular tumours, which can develop in patients with congenital adrenal hyperplasia. If left untreated, they can cause testicular tissue damage and infertility. Ultrasound is the imaging modality of choice allowing for non-invasive diagnosis provided that characteristic features are seen. In recent times, magnetic resonance imaging characteristics have also been described to aid diagnosis. CASE REPORT: This case describes the imaging features of multiple bilateral TART in a young patient with congenital adrenal hyperplasia. Traditional greyscale and colour Doppler ultrasound demonstrated intratesticular, predominantly hypoechoic areas, with increased surrounding colour Doppler flow. In addition, contrast-enhanced ultrasound and strain elastography were performed, showing increased TART vascularisation and increased stiffness in the hypoechoic areas. Subsequent magnetic resonance imaging confirmed bilateral lobulated, well demarcated, intratesticular lesions, which appeared predominantly isointense on T1-weighted imaging, hypointense on T2-weighted imaging, with heterogeneous enhancement following gadolinium administration. DISCUSSION: This case illustrates the sonographic features including greyscale, colour Doppler, contrast and elastography ultrasound of TART in a patient with congenital adrenal hyperplasia. Both contrast-enhanced ultrasound and elastography can provide information about tissue properties not normally derived from conventional ultrasound and aid accurate diagnosis. Additional magnetic resonance imaging is not normally required with typical ultrasound appearances. The unusual findings seen on the ultrasound examination were likely secondary to poor compliance with treatment. CONCLUSION: Familiarity and recognition of characteristic and uncommon imaging features of these tumours are important to avoid misdiagnosis and surgical intervention.

Characterizing stochastic cell-cycle dynamics in exponential growth.

Two powerful and complementary experimental approaches are commonly used to study the cell cycle and cell biology: One class of experiments characterizes the statistics (or demographics) of an unsynchronized exponentially growing population, while the other captures cell-cycle dynamics, either by time-lapse imaging of full cell cycles or in bulk experiments on synchronized populations. In this paper, we study the subtle relationship between observations in these two distinct experimental approaches. We begin with an existing model: A single-cell deterministic description of cell-cycle dynamics where cell states (i.e., periods or phases) have precise lifetimes. We then generalize this description to a stochastic model in which the states have stochastic lifetimes, as described by arbitrary probability distribution functions. Our analyses of the demographics of an exponential culture reveal a simple and exact correspondence between the deterministic and stochastic models: The corresponding state ages in the deterministic model are equal to the exponential mean of the age in the stochastic model. An important implication is therefore that the demographics of an exponential culture will be well fit by a deterministic model even if the state timing is stochastic. Although we explore the implications of the models in the context of the Escherichia coli cell cycle, we expect both the models as well as the significance of the exponential-mean lifetimes to find many applications in the quantitative analysis of cell-cycle dynamics in other biological systems.

Pan-SARS neutralizing responses after third boost vaccination in non-human primate immunogenicity model.

The emergence of SARS-CoV-2 variants, especially Beta and Delta, has raised concerns about the reduced protection from previous infection or vaccination based on the original Wuhan-Hu-1 (D614) virus. To identify promising regimens for inducing neutralizing titers towards new variants, we evaluated monovalent and bivalent mRNA vaccines either as primary vaccination or as a booster in nonhuman primates (NHPs). Two mRNA vaccines, D614-based MRT5500 and Beta-based MRT5500ß, tested in sequential regimens or as a bivalent combination in naïve NHPs produced modest neutralizing titers to heterologous variants. However, when mRNA vaccines were administered as a booster to pre-immune NHPs, we observed a robust increase in neutralizing titers with expanded breadth towards all tested variants, and notably SARS-CoV-1. The breadth of the neutralizing response was independent of vaccine sequence or modality, as we further showed either MRT5500 or recombinant subunit Spike protein (with adjuvant) can serve as boosters to induce broadly neutralizing antibodies in the NHPs primed with MRT5500. The data support the notion that a third vaccination is key to boosting existing titers and improving the breadth of antibodies to address variants of concern, including those with an E484K mutation in the Receptor Binding Domain (RBD) (Beta, Gamma).

Outcomes after Emergency Admission with a Diabetic Foot Attack Indicate a High Rate of Healing and Limb Salvage But Increased Mortality: 18-Month Follow-up Study.

AIM: The diabetic foot attack (DFA) is perhaps the most devastating form of diabetic foot infection, presenting with rapidly progressive skin and tissue necrosis, threatening both limb and life. However, clinical outcome data in this specific group of patients are not available. METHODS: Analysis of 106 consecutive patients who underwent emergency hospitalisation for DFA (TEXAS Grade 3B or 3D and Infectious Diseases Society of America (IDSA) Class 4 criteria). Outcomes evaluated were: 1) Healing 2) major amputation 3) death 4) not healed. The first outcome reached in one of these four categories over the follow-up period (18.4±3.6 months) was considered. We also estimated amputation free survival. RESULTS: Overall, 57.5% (n=61) healed, 5.6% (n=6) underwent major amputation, 23.5% (n=25) died without healing and 13.2% (n=14) were alive without healing. Predictive factors associated with outcomes were: Healing (age<60, p=0.0017; no Peripheral arterial disease (PAD) p= 0.002; not on dialysis p=0.006); major amputation (CRP>100 mg/L, p=0.001; gram+ve organisms, p=0.0013; dialysis, p= 0.001), and for death (age>60, p= 0.0001; gram+ve organisms p=0.004; presence of PAD, p=0.0032; CRP, p=0.034). The major amputation free survival was 71% during the first 12 months from admission, however it had reduced to 55.4% by the end of the follow-up period. CONCLUSIONS: In a unique population of hospitalised individuals with DFA, we report excellent healing and limb salvage rates using a dedicated protocol in a multidisciplinary setting. An additional novel finding was the concerning observation that such an admission was associated with high 18-month mortality, almost all of which was after discharge from hospital.

Comparison of Chicken Immune Responses after Inoculation with H5 Avian Influenza Virus-like Particles Produced by Insect Cells or Pupae.

INTRODUCTION: Novel clade 2.3.4.4 H5 highly pathogenic avian influenza virus (HPAIV) outbreaks have occurred since early 2015 in Taiwan and impacted the island economically, like they have many countries. This research investigates the immunogenicity of two HPAIV-like particles to assess their promise as vaccine candidates. MATERIAL AND METHODS: The haemagglutinin (HA) gene derived from clade 2.3.4.4 H5 HPAIV and matrix protein 1 (M1) gene were cloned into the pFastBac Dual baculovirus vector. The resulting recombinant viruses were expressed in Spodoptera frugiperda moth (Sf)21 cells and silkworm pupae to generate Sf21 virus-like particles (VLP) and silkworm pupa VLP. Two-week-old specific pathogen-free chickens were immunised and their humoral and cellular immune responses were analysed. RESULTS: The silkworm pupa VLP had higher haemagglutination competence. Both VLP types elicited haemagglutination inhibition antibodies, anti-HA antibodies, splenic interferon gamma (IFN-γ) and interleukin 4 (IL-4) mRNA expression, and CD4+/CD8+ ratio elevation. However, chickens receiving silkworm pupa VLP exhibited a significantly higher anti-HA antibody titre in ELISA after vaccination. Although Sf21 VLP recipients expressed more IFN-γ and IL-4, the increase in IFN-γ did not significantly raise the CD4+/CD8+ ratio and the increase in IL-4 did not promote anti-HA antibodies. CONCLUSION: Both VLP systems possess desirable immunogenicity in vivo. However, in respect of immunogenic efficacy and the production cost, pupa VLP may be the superior vaccine candidate against clade 2.3.4.4 H5 HPAIV infection.

Contrast-enhanced ultrasound in pediatric interventional radiology.

There is growing interest in the use of contrast-enhanced ultrasound (CEUS) in diagnostic and interventional radiology. CEUS applications in interventional radiology are performed with intravascular or intracavitary administration of microbubble-based US contrast agents to allow for real-time evaluation of their distribution within the vascular bed or in body cavities, respectively, providing additional information beyond gray-scale US alone. The most common interventional-radiology-related CEUS applications in children have been extrapolated from those in adults, and they include the use of CEUS to guide lesion biopsy and to confirm drain placement in pleural effusions and intra-abdominal fluid collections. Other applications are emerging in interventional radiology for use in adults and children, including CEUS to optimize sclerotherapy of vascular malformations, to guide arthrography, and for lymphatic interventions. In this review article we present a wide range of interventional-radiology-related CEUS applications, emphasizing the current and potential uses in children. We highlight the technical parameters of the CEUS examination and discuss the main imaging findings.