Invited review: Modeling milk stability.

Novel insights into the stability of milk and milk products during storage and processing result from describing caseins near neutral pH as hydrophilic, intrinsically disordered, proteins. Casein solubility is strongly influenced by pH and multivalent ion binding. Solubility is high at neutral pH or above but decreases as casein net charge approaches zero, allowing a condensed casein phase or gel to form then increases at lower pH. Of particular importance for casein micelle stability near neutral pH is the proportion of free caseins in the micelle (i.e., caseins not bound directly to nanoclusters of calcium phosphate). Free caseins are more soluble and better able to act as molecular chaperones (to prevent casein and whey protein aggregation) than bound caseins. Some free caseins are highly phosphorylated and can also act as mineral chaperones to inhibit the growth of calcium phosphate phases and prevent mineralized deposits from forming on membranes or heat exchangers. Thus, casein micelle stability is reduced when free caseins bind to amyloid fibrils, destabilized whey proteins or calcium phosphate. The multivalent-binding model of the casein micelle quantitatively describes these and other factors affecting the stability of milk and milk protein products during manufacture and storage.

Recruitment to higher specialty training in anaesthesia in the UK during the COVID-19 pandemic: a national survey.

There were more applications for higher specialty training posts in anaesthesia in the UK starting in August 2021 than in previous years, with approximately two-thirds being unsuccessful. We surveyed applicants to investigate their experience of the recruitment process (response rate 536/1056; 51%). Approximately 61% of respondents were not offered ST3 posts (n = 326). We enquired about their career plans for the next 12-24 months. Most respondents (79%) intended to take up a post equivalent to a third year of core training or a clinical fellow post from August 2021. Other options considered included: pursuing work abroad (17%); embarking on career breaks (16%); taking up higher training posts in intensive care medicine (15%); and permanently leaving medicine (9%). Nine per cent of respondents also expressed plans to pursue training in another medical specialty. Some expressed an intention to pursue further education or research (10%). A large proportion (42%) expressed a lack of confidence in being able to achieve the training requirements to later apply for a higher training post. The majority reported not feeling confident in achieving specialist registration in anaesthesia in the future without a training number (75%), and noted disruption to their wider life plans from the impending time out of training (78%). Sentiment analysis of free-text responses indicated generally negative sentiment about the recruitment process. Themes elicited included: feeling the recruitment process was unfair; burnout and negative impact on well-being; difficulties in making life plans; and feeling undervalued and abandoned. These results suggest that junior anaesthetic doctors in the UK negatively perceived postgraduate training structures and changes to the postgraduate curriculum and experienced difficulties in securing higher training.

Circulating lectin pathway proteins do not predict short-term cardiac outcomes after myocardial infarction.

Despite improvements in treatment, coronary artery disease is still responsible for one-third of all deaths globally, due predominantly to myocardial infarction (MI) and stroke. There is an important potential in developing new strategies for treatment of patients with these conditions. Inflammation, and in particular the actions of the complement system, has emerged as part of the pathogenesis in reperfusion injury in patients with MI. To further qualify this, we examined the association between the plasma levels of lectin pathway proteins and myocardial end-points, left ventricular ejection fraction (LVEF) and infarct size in a cohort of patients with ST-elevation myocardial infarction (STEMI). A blood sample was drawn the day after percutaneous coronary intervention from 73 patients with STEMI. The primary end-points, LVEF and infarct size, were measured with magnetic resonance imaging 6-9 days after the infarct. Complement pattern-recognition molecules of the lectin pathway (mannan-binding lectin, H-ficolin, L-ficolin and M-ficolin) were analysed along with soluble membrane attack complex (sMAC) and C-reactive protein (CRP) in plasma with immunofluorometric assays <50%. CRP correlated negatively with LVEF, regression coefficient = -0·17 (P = 0·01). None of the lectin pathway proteins correlated to LVEF or infarct size, nor did soluble membrane attack complex (sMAC). There were no differences in plasma levels of these complement proteins when comparing patients with ejection fraction <50% to patients with ejection fraction <50%. Pattern-recognition molecules of the lectin pathway and sMAC do not predict short-term cardiac outcomes after MI.

Plasma levels of MASP-1, MASP-3 and MAp44 in patients with type 2 diabetes: influence of glycaemic control, body composition and polymorphisms in the MASP1 gene.

Mounting evidence indicates that adverse activation of the complement system plays a role in the development of diabetic vascular complications. Plasma levels of the complement proteins mannan-binding lectin (MBL) and its associated serine proteases (MASP-1 and MASP-2) are elevated in diabetes. We hypothesized that single nucleotide polymorphisms (SNPs) in the MASP1 gene may contribute to altered plasma levels of the belonging gene products; MASP-1, MASP-3 and mannan-binding lectin-associated protein of 44 kDa (MAp44) in patients with type 2 diabetes. To investigate this, we compared plasma levels of MASP-1, MASP-3 and MAp44 in 100 patients with type 2 diabetes and 100 sex- and age-matched controls. Ten carefully selected SNPs were analysed using TaqMan® genotyping assay. Additionally, we included a streptozotocin-induced diabetes mouse model to directly examine the effect of inducing diabetes on MASP-1 levels. MASP-1 levels were significantly higher among patients with type 2 diabetes compared with healthy controls (P = 0·017). Five SNPs (rs874603, rs72549254, rs3774275, rs67143992, rs850312) in the MASP1 gene were associated with plasma levels of MASP-1, MASP-3 and MAp44. In the diabetes mouse model, diabetic mice had significantly higher MASP-1 levels than control mice (P = 0·003). In conclusion, MASP-1 levels were higher among patients with type 2 diabetes and diabetic mice. The mechanism behind this increase remains elusive.

Tirzepatide attenuates mammary tumor progression in diet-induced obese mice.

We report for the first time an anticancer benefit of tirzepatide-a dual glucagon-like peptide 1 and glucose-dependent insulinotropic polypeptide receptor agonist-in a model of obesity and breast cancer in female mice. Long-term tirzepatide treatment induced weight loss, mitigated obesity-driven changes in circulating metabolic hormone levels, and suppressed orthotopic E0771 mammary tumor growth. Relative to tirzepatide, chronic calorie restriction, an established anticancer intervention in preclinical models, promoted even greater weight loss, systemic hormonal regulation, and tumor suppression. We conclude that tirzepatide represents a promising pharmacologic approach for mitigating the procancer effects of obesity. Moreover, strategies promoting greater weight loss than achieved with tirzepatide alone may augment the anticancer benefits of tirzepatide.

Invited review: Caseins and the casein micelle: their biological functions, structures, and behavior in foods.

A typical casein micelle contains thousands of casein molecules, most of which form thermodynamically stable complexes with nanoclusters of amorphous calcium phosphate. Like many other unfolded proteins, caseins have an actual or potential tendency to assemble into toxic amyloid fibrils, particularly at the high concentrations found in milk. Fibrils do not form in milk because an alternative aggregation pathway is followed that results in formation of the casein micelle. As a result of forming micelles, nutritious milk can be secreted and stored without causing either pathological calcification or amyloidosis of the mother's mammary tissue. The ability to sequester nanoclusters of amorphous calcium phosphate in a stable complex is not unique to caseins. It has been demonstrated using a number of noncasein secreted phosphoproteins and may be of general physiological importance in preventing calcification of other biofluids and soft tissues. Thus, competent noncasein phosphoproteins have similar patterns of phosphorylation and the same type of flexible, unfolded conformation as caseins. The ability to suppress amyloid fibril formation by forming an alternative amorphous aggregate is also not unique to caseins and underlies the action of molecular chaperones such as the small heat-shock proteins. The open structure of the protein matrix of casein micelles is fragile and easily perturbed by changes in its environment. Perturbations can cause the polypeptide chains to segregate into regions of greater and lesser density. As a result, the reliable determination of the native structure of casein micelles continues to be extremely challenging. The biological functions of caseins, such as their chaperone activity, are determined by their composition and flexible conformation and by how the casein polypeptide chains interact with each other. These same properties determine how caseins behave in the manufacture of many dairy products and how they can be used as functional ingredients in other foods.

Darwinian transformation of a 'scarcely nutritious fluid' into milk.

In an early challenge to an aspect of Darwin's theory of natural selection, Jackson Mivart contended that milk could not have evolved 'from a scarcely nutritious fluid from an accidentally hypertrophied cutaneous gland'. The evolutionary change from a gland secretion to milk involves an increase in calcium and protein concentrations by up to 100- and 1000-fold, respectively. Even so, the challenge, we suggest, is not just a problem of scale. An increase in the concentrations of calcium and phosphate brings an increased risk of calcification of the secretory gland because calcium phosphate is highly insoluble. In addition, two of the four constituent milk casein proteins (κ and α(S2)) aggregate to produce toxic amyloid fibrils. It is proposed that both problems were solved through the cosecretion of ancestral ß- and κ-caseins to form a stable amorphous aggregate of both proteins with sequestered amorphous calcium phosphate, that is, a primordial casein micelle. Evolutionarily, a gradual increase in the concentration of casein micelles could therefore produce progressively more nutritious fluids for the neonate without endangering the reproductive potential of the mother.

Assessing functional recovery following total knee replacement surgery using objective classification of level gait data and patient-reported outcome measures.

BACKGROUND: Patient recovery can be quantified objectively, via gait analysis, or subjectively, using patient reported outcome measures. Association between these measures would explain the level of disability reported in patient reported outcome measures and could assist with therapeutic decisions. METHODS: Total knee replacement outcome was assessed using objective classification and patient-reported outcome measures (Knee Outcome Survey and Oxford Knee Scores). A classifier was trained to distinguish between healthy and osteoarthritic characteristics using knee kinematics, ground reaction force and temporal gait data, combined with anthropometric data from 32 healthy and 32 osteoarthritis knees. For the osteoarthritic cohort, classification of 20 subjects quantified changes at up to 3 timepoints post-surgery. FINDINGS: Osteoarthritic classification was reduced for 17 subjects when comparing pre- to post-operative assessments, however only 6 participants achieved non-pathological classification and only 4 of these were classified as non-pathological at 12 months. In 15 cases, the level of osteoarthritic classification did not decrease between every post-operative assessment. For an individual's recovery, classification outputs correlated (r > 0.5) with knee outcome survey for 75% of patients and oxford knee score for 78% of patients (based on 20 and 9 subjects respectively). Classifier outputs from all visits of the combined total knee replacement sample correlated moderately with knee outcome survey (r > 0.4) and strongly with oxford knee score (r > 0.6). INTERPRETATION: Biomechanical deficits existed in most subjects despite improvements in Patient Reported Outcome Measures, with larger changes reported subjectively as compared to measured objectively. Objective Classification provides additional insight alongside Patient Reported Outcomes when reporting recovered outcomes.

Microbiomes of microscopic marine invertebrates do not reveal signatures of phylosymbiosis.

Animals and microorganisms often establish close ecological relationships. However, much of our knowledge about animal microbiomes comes from two deeply studied groups: vertebrates and arthropods. To understand interactions on a broader scale of diversity, we characterized the bacterial microbiomes of close to 1,000 microscopic marine invertebrates from 21 phyla, spanning most of the remaining tree of metazoans. Samples were collected from five temperate and tropical locations covering three marine habitats (sediment, water column and intertidal macroalgae) and bacterial microbiomes were characterized using 16S ribosomal RNA gene sequencing. Our data show that, despite their size, these animals harbour bacterial communities that differ from those in the surrounding environment. Distantly related but coexisting invertebrates tend to share many of the same bacteria, suggesting that guilds of microorganisms preferentially associated with animals, but not tied to any specific host lineage, are the main drivers of the ecological relationship. Host identity is a minor factor shaping these microbiomes, which do not show the same correlation with host phylogeny, or 'phylosymbiosis', observed in many large animals. Hence, the current debate on the varying strength of phylosymbiosis within selected lineages should be reframed to account for the possibility that such a pattern might be the exception rather than the rule.

A seafood risk tool for assessing and mitigating chemical and pathogen hazards in the aquaculture supply chain.

Intricate links between aquatic animals and their environment expose them to chemical and pathogenic hazards, which can disrupt seafood supply. Here we outline a risk schema for assessing potential impacts of chemical and microbial hazards on discrete subsectors of aquaculture-and control measures that may protect supply. As national governments develop strategies to achieve volumetric expansion in seafood production from aquaculture to meet increasing demand, we propose an urgent need for simultaneous focus on controlling those hazards that limit its production, harvesting, processing, trade and safe consumption. Policies aligning national and international water quality control measures for minimizing interaction with, and impact of, hazards on seafood supply will be critical as consumers increasingly rely on the aquaculture sector to supply safe, nutritious and healthy diets.

Posttransplantation hepatitis B prophylaxis with combination oral nucleoside and nucleotide analog therapy.

Liver transplant recipients are at risk of developing recurrent hepatitis B after liver transplantation for hepatitis B virus (HBV)-related liver disease. We evaluated the efficacy of a new hepatitis B prophylaxis regimen involving conversion from at least 12 months of HBIg with lamivudine to combination therapy with an oral nucleoside and nucleotide analog. Between June 2008 and May 2010, a total of 61 liver transplant recipients were converted to a combination of a nucleoside and nucleotide analog. The mean (±standard deviation) follow-up time after conversion was 15.0 (±6.1) months. Recurrent HBV occurred in two (3.3%) patients at 3.1 and 16.6 months after HBIg cessation. The overall person time incidence rate for HBV recurrence after HBIg cessation was 2.7 cases per 100 person-years. The estimate of HBV recurrence was 1.7% at 1 year after HBIg cessation. HBIg cessation a minimum of 12 months after liver transplantation with subsequent combination therapy with a nucleoside and nucleotide analog provides effective prophylaxis against recurrent HBV infection. The clinical implications of HBsAg detection without clinical, biochemical or molecular manifestations of recurrent hepatitis B require further study.

Decrease in cardiac catheterization and MI during COVID pandemic.

The consequences of severe acute viral respiratory syndrome (COVID 19) pandemic include collateral effects, one of which has been the significant reduction in routine hospital work. With widespread reports indicating reduction of cardiac procedures including MI presentation to hospitals, we aimed to analyze the local data over a 10-week period during lockdown in a tertiary cardiac centre Catheter Laboratory in England. METHODS: We conducted a retrospective review of the coronary catheterisation procedures and admissions with MI over the peak COVID-19 pandemic 10-week period (23rd March-30th May) in 2020, compared with the same 10-week period (25th March-2nd June) in 2019. RESULTS: In 2019, 539 patients were admitted to the Cath lab for coronary catheterisation (M = 385:F = 154; mean age 65 years; STEMI = 186, NSTEMI = 192, elective = 161). In 2020, during peak period of COVID19 pandemic in England, a total of 278 patients were admitted for coronary catheterisation over the 10-week period (M = 201:F = 77; mean age 60.5 years; STEMI = 132, NSTEMI = 118, elective = 28). During peak COVID19 pandemic, this represents a 48.4% drop in all coronary catheterisations. The reduction in STEMI was 29% (54 less), in NSTEMI was 38.9% (74 less) and elective procedures dropped by 83% (133 less). CONCLUSION: During peak COVID hospital admission period in England, we report a 48.5% reduction in coronary catheterisation in our tertiary hospital. These results are consistent with reports from other countries, and highlight the worrying potential consequences for these patients arising from delays in presentation with MI, and the challenges for restoring services post-pandemic.

Friend virus-specific cytotoxic T lymphocytes recognize both gag and env gene-encoded specificities.

We have constructed a series of "synthetic" target cell lines for an analysis of the specificity of anti-Friend virus (FV) CTL. Our results show that murine H-2 genes and individual retroviral genes can be stable expressed in Fisher rat embryo (FRE) cells, and that their products have the potential to form target structures recognized by mouse CTL. Cells expressing H-2Db and either the env or gag genes of one component of FV, helper Friend murine leukemia virus (FMuLV), were lysed by anti-FV CTL and by CTL generated against FMuLV alone. Experiments with Db-transfected FRE clones infected only with the replication-defective spleen focus-forming virus (SFFV) component of FV indicate that the SFFV genome also provides specificities recognized by both anti-FV and anti-FMuLV CTL, thus demonstrating the existence of a crossreactive CTL population. An unexpected finding was that anti-FMuLV CTL, but not anti-FV CTL were also able to lyse FRE clones that expressed H-2Kb in either the presence or absence of FV. The use of heterologous cell lines for the construction of synthetic target cells thus offers a useful approach for the analysis of T cell specificity.

FBL-reactive CD8+ cytotoxic and CD4+ helper T lymphocytes recognize distinct Friend murine leukemia virus-encoded antigens.

Immunization of C57BL/6 (B6) mice with FBL, a Friend murine leukemia virus (F-MuLV), induces both tumor-specific cytolytic CD8+ (CTL) and lymphokine-producing CD4+ Th that are effective in adoptive therapy of B6 mice bearing disseminated FBL leukemia. The current study evaluated the F-MuLV antigenic determinants expressed on FBL that are recognized by FBL-reactive CD8+ and CD4+ T cells. To identify the specificity of the FBL-reactive CD8+ CTL, Fisher rat embryo fibroblast (FRE) cells transfected with plasmids encoding F-MuLV gag or envelope (env) gene products plus the class I-restricting element Db were utilized. FBL-reactive CTL recognized FRE target cells transfected with the F-MuLV gag-encoded gene products, but failed to recognize targets expressing F-MuLV env. Attempts to generate env-specific CD8+ CTL by immunization with a recombinant vaccinia virus containing an inserted F-MuLV env gene were unsuccessful, despite the generation of a cytolytic response to vaccinia epitopes, implying that B6 mice fail to generate CD8+ CTL to env determinants. By contrast, CD4+ Th clones recognized FRE target cells transfected with env and not gag genes, and immunization with the recombinant vaccinia virus induced an env-specific CD4+ T cell response. These data show that in a Friend retrovirus-induced tumor model in which tumor rejection can be mediated by either CTL or Th, antigens derived from discrete retroviral proteins are predominantly responsible for activation of each T cell subset.

Does the choice of stair gait cycle affect resulting knee joint kinematics and moments?

Stair gait is a useful activity for the assessment of knee function. The aim of this study was to determine whether knee joint kinematics and moments are affected by the choice of stair gait cycle (SGC) and the step used to measure ground reaction forces (GRFs). This was investigated through motion analysis of ten non-pathological subjects as they ascended and descended a four-step staircase. The SGCs compared for ascent were, first, step 1 (measuring GRFs) to step 3 and, second, step 2 (measuring GRFs) to step 4, and vice versa for stair descent. Knee joint kinematics were not significantly influenced by the choice of SGC. For ascent, significantly larger peak adduction moments were measured for SGCs beginning on step 1 (0.30 +/- 0.08 N m/kg) than for SGCs beginning on step 2 (0.23 +/- 0.09 N m/kg). For descent, the second flexion moment peak was found to be significantly larger for SGCs ending on step 2 (1.17 +/- 0.25 N m/kg) than for SGCs ending on step 1 (0.97 +/- 0.19 N m/kg), and the first adduction moment peak was found to be significantly larger for SGCs ending on step 2 (0.28 +/- 0.15 N m/kg) than for SGCs ending on step 1 (0.21 +/- 0.18 N m/kg). This study highlights important considerations when planning stair gait measurement protocols and comparing results from studies made by other laboratories.

Challenges facing quantification of rat locomotion along beams of varying widths.

Optoelectronic motion capture systems have been widely used to investigate temporal gait parameters in humans and animals in order to understand function and behavioural attributes of different pathologies, e.g. Parkinson's disease (PD). The aim of the present paper was to investigate the practicality of utilising this system to investigate the effects of a unilateral 6-hydroxydopamine (6-OHDA) lesion on rat locomotion while walking on beams of varying widths (graduated, narrow, and wide). Temporal gait parameters of ten male Lister Hooded rats (five controls and five hemiparkinsonian) were observed using passive markers placed in locations that were representative of their four limbs and their body axis. The results demonstrate that marker-based motion capture can provide an effective and simple approach to quantifying temporal gait parameters for rat models of PD. They also reveal how the width of the path affects the locomotion in both experimental cohorts. Such measurements can be compared with human motion analysis to explore correlations between the animal model and human behaviour, which is an important step for translational medicine.

Cell-cycle-phase progression analysis identifies unique phenotypes of major prognostic and predictive significance in breast cancer.

Multiparameter analysis of core regulatory proteins involved in G1-S and G2-M cell-cycle transitions provides a powerful biomarker readout for assessment of the cell-cycle state. We have applied this algorithm to breast cancer to investigate how the cell cycle impacts on disease progression. Protein expression profiles of key constituents of the DNA replication licensing pathway (Mcm2, geminin) and mitotic machinery (Plk1, Aurora A and the Aurora substrate histone H3S10ph) were generated for a cohort of 182 patients and linked to clinicopathological parameters. Arrested differentiation and genomic instability were associated with an increased engagement of cells into the cell division cycle (P<0.0001). Three unique cell-cycle phenotypes were identified: (1) well-differentiated tumours composed predominantly of Mcm2-negative cells, indicative of an out-of-cycle state (18% of cases); (2) high Mcm2-expressing tumours but with low geminin, Aurora A, Plk1 and H3S10ph levels (S-G2-M progression markers), indicative of a G1-delayed/arrested state (24% cases); and (3) high Mcm2-expressing tumours and also expressing high levels of the S-G2-M progression markers, indicative of accelerated cell-cycle progression (58% of cases). The active cell-cycle progression phenotype had a higher risk of relapse when compared with out-of-cycle and G1-delayed/arrested phenotypes (HR=3.90 (1.81-8.40, P<0.001)), and was associated with Her-2 and triple negative subtypes (P<0.001). It is of note that high-grade tumours with the G1-delayed/arrested phenotype showed an identical low risk of relapse compared with well-differentiated out-of-cycle tumours (HR=1.00 (0.22-4.46), P=0.99). Our biomarker algorithm provides novel insights into the cell-cycle state of dynamic tumour cell populations in vivo. This information is of major prognostic significance and may impact on individualised therapeutic decisions. Patients with an accelerated phenotype are more likely to derive benefit from S- and M-phase-directed chemotherapeutic agents.

Growth factors: a role in guiding axons?

A remarkable finding to emerge in recent years is that the early brain neuroepithelium is highly patterned before axonogenesis begins. Growth factors are among a variety of classes of molecules whose regionalized expression divides the early brain into molecularly distinct domains. Thus, when axons first grow to their synaptic targets, growth factor signalling may help them to navigate. This review discusses recent studies that reveal that growth factors can act as chemoattractants and repellents and that growth factor signalling is important for target entry. These new findings raise the compelling idea that growth factors play an active role in axon navigation.

Minor components of the DNA of Physarum polycephalum. Cellular location and metabolism.

DNA metabolism in the slime mold Physarum polycephalum was studied by centrifugation in CsCl of lysates of cultures labeled with radioactive thymidine at various times in the cell cycle. During the G(2) (premitotic) phase of the cell cycle, two components of the DNA are labeled. One component is lighter (buoyant density 1.686 g/cc) than the mean of the principal DNA (1.700 g/cc), and one is heavier (approximately 1.706 g/cc). The labeled light DNA was identified chemically by its denaturability, its susceptibility to DNase, and the recovery of its radioactivity in thymine. Cell fractionation studies showed that the heavy and the principal DNA components are located in the nucleus and that the light DNA is in the cytoplasm. The light DNA comprises approximately 10% of the DNA. About (1/3)-(1/2) of the light DNA is synthesized during the S period, and the remainder is synthesized throughout G(2) (there is no G(1) in Physarum). The light DNA is metabolically stable. A low, variable level of incorporation of radioactive thymidine into the principal, nuclear DNA component was observed during G(2).

A carrot leucine-rich-repeat protein that inhibits ice recrystallization.

Many organisms adapted to live at subzero temperatures express antifreeze proteins that improve their tolerance to freezing. Although structurally diverse, all antifreeze proteins interact with ice surfaces, depress the freezing temperature of aqueous solutions, and inhibit ice crystal growth. A protein purified from carrot shares these functional features with antifreeze proteins of fish. Expression of the carrot complementary DNA in tobacco resulted in the accumulation of antifreeze activity in the apoplast of plants grown at greenhouse temperatures. The sequence of carrot antifreeze protein is similar to that of polygalacturonase inhibitor proteins and contains leucine-rich repeats.