Geographic disparities in thyroid cancer staging at presentation: Insights from an Australian context.

BACKGROUND: Thyroid cancer diagnoses have increased over recent decades at a rate much higher than that of any other cancer in Australia. Rural patients are known to have reduced access to healthcare and may have different thyroid cancer presentation rates. This study examined the relationship between thyroid cancer diagnosis and patient rurality. METHODS: Data from the Australia and New Zealand Thyroid Cancer Registry from 2017 to 2022 were analyzed, stratifying patient postcodes into rurality groups using the Australian Statistical Geography Standard. The American Thyroid Association (ATA) guidelines were used to stratify risk categories and management to compare treatment adequacy between the groups. Statistical analysis assessed demographic, clinical, and management differences. RESULTS: Among 1766 patients, 70.6% were metropolitan (metro) and 29.4% were non-metropolitan (non-metro). Non-metro patients were older at diagnosis (median 56 vs. 50 years, p < 0.001), presented more frequently with T stage greater than 1 (stage 2-4, 41.9% vs. 34.8%, and p = 0.005), AJCC stage greater than 1 (stage 2-4, 18.5% vs. 14.6%, and p = 0.019), and cancers larger than 4 cm (14.3% vs. 9.9%, p = 0.005). No significant differences in treatment adequacy were observed between the groups for ATA low-risk cancers. CONCLUSIONS: Non-metropolitan patients in the registry present with more advanced thyroid cancer, possibly due to differences in healthcare access. Further research should assess long-term survival outcomes and influencing factors. Understanding the impact on patient outcomes and addressing healthcare access barriers can optimize thyroid cancer care across geographic regions in Australia.

A secreted bacterial protein protects bacteria from cationic antimicrobial peptides by entrapment in phase-separated droplets.

Mammalian hosts combat bacterial infections through the production of defensive cationic antimicrobial peptides (CAPs). These immune factors are capable of directly killing bacterial invaders; however, many pathogens have evolved resistance evasion mechanisms such as cell surface modification, CAP sequestration, degradation, or efflux. We have discovered that several pathogenic and commensal proteobacteria, including the urgent human threat Neisseria gonorrhoeae, secrete a protein (lactoferrin-binding protein B, LbpB) that contains a low-complexity anionic domain capable of inhibiting the antimicrobial activity of host CAPs. This study focuses on a cattle pathogen, Moraxella bovis, that expresses the largest anionic domain of the LbpB homologs. We used an exhaustive biophysical approach employing circular dichroism, biolayer interferometry, cross-linking mass spectrometry, microscopy, size-exclusion chromatography with multi-angle light scattering coupled to small-angle X-ray scattering (SEC-MALS-SAXS), and NMR to understand the mechanisms of LbpB-mediated protection against CAPs. We found that the anionic domain of this LbpB displays an α-helical secondary structure but lacks a rigid tertiary fold. The addition of antimicrobial peptides derived from lactoferrin (i.e. lactoferricin) to the anionic domain of LbpB or full-length LbpB results in the formation of phase-separated droplets of LbpB together with the antimicrobial peptides. The droplets displayed a low rate of diffusion, suggesting that CAPs become trapped inside and are no longer able to kill bacteria. Our data suggest that pathogens, like M. bovis, leverage anionic intrinsically disordered domains for the broad recognition and neutralization of antimicrobials via the formation of biomolecular condensates.

Evolving challenges of leadership in surgery to improve inclusivity, representation, and well-being.

BACKGROUND: Leadership is a complex and demanding process crucial to maintaining quality in surgical systems of care. Once an autocratic practice, modern-day surgical leaders must demonstrate inclusivity, flexibility, emotional competence, team-building, and a multidisciplinary approach. The complex healthcare environment challenges those in leadership positions. The aim of this narrative review was to consolidate the major challenges facing surgeons today and to suggest evidence-based strategies to support surgical leaders. METHODS: Google Scholar, PubMed, MEDLINE, and Ovid databases were searched to review literature on the challenges faced by surgical leaders. The commonly identified areas that compromise inclusivity and productive leadership practices were consolidated into 10 main subheadings. Further research was conducted using the aforementioned databases to outline the importance of addressing such challenges, and to consolidate evidence-based strategies to resolve them. RESULTS: The importance of increasing representation of marginalized groups in leadership positions, including women, ethnic groups, the queer community, and ageing professionals, has been identified by surgical colleges in many countries. Leaders must create a collegial environment with proactive, honest communication and robust reporting pathways for victims of workplace harassment. The retention of diverse, empowering, and educating leaders relies on equitable opportunities, salaries, recognition, and support. Thus, it is important to implement formal training and mentorship, burnout prevention, conflict management, and well-being advocacy. CONCLUSION: There are two aspects to addressing challenges facing surgical leadership; improving advocacy by and for leaders. Systems must be designed to support surgical leaders through formal education and training, meaningful mentorship programmes, and well-being advocacy, thus enabling them to proactively and productively advocate and care for their patients, colleagues, and professional communities.

Zinc acquisition and its contribution to Klebsiella pneumoniae virulence.

Klebsiella pneumoniae is a World Health Organization priority pathogen and a significant clinical concern for infections of the respiratory and urinary tracts due to widespread and increasing resistance to antimicrobials. In the absence of a vaccine, there is an urgent need to identify novel targets for therapeutic development. Bacterial pathogens, including K. pneumoniae, require the d-block metal ion zinc as an essential micronutrient, which serves as a cofactor for ~6% of the proteome. During infection, zinc acquisition necessitates the use of high affinity uptake systems to overcome niche-specific zinc limitation and host-mediated nutritional immunity. Here, we report the identification of ZnuCBA and ZniCBA, two ATP-binding cassette permeases that are highly conserved in Klebsiella species and contribute to K. pneumoniae AJ218 zinc homeostasis, and the high-resolution structure of the zinc-recruiting solute-binding protein ZniA. The Znu and Zni permeases appear functionally redundant with abrogation of both systems required to reduce K. pneumoniae zinc accumulation. Disruption of both systems also exerted pleiotropic effects on the homeostasis of other d-block elements. Zinc limitation perturbed K. pneumoniae cell morphology and compromised resistance to stressors, such as salt and oxidative stress. The mutant strain lacking both systems showed significantly impaired virulence in acute lung infection models, highlighting the necessity of zinc acquisition in the virulence and pathogenicity of K. pneumoniae.

The Use of Five-Strand Hamstring Autograft to Increase the Graft Size in Anterior Cruciate Ligament Reconstruction-A Prospective Cohort Study With Satisfactory Early Clinical Results.

Objectives: To report on the outcomes of using 5-strand hamstring autograft to increase the graft size for anterior cruciate ligament (ACL) reconstruction and to determine whether the clinical results are comparable to using conventional 4-strand graft. Methods: A prospective cohort study of patients with arthroscopic-assisted single-bundle ACL reconstruction using hamstring autograft from January 2019 to June 2021.The patients were prospectively recruited to undergo ACL reconstruction with either 5-strand hamstring graft (group A) or 4-strand hamstring graft (group B). Results: In total, 45 patients were included into the study. The mean diameter of the final graft was 8.9 ± 0.6 cm in the 5-strand group and 7.5 ± 0.8 cm in the 4-strand group (P < .001). Four-strand graft diameter measurements were taken intraoperatively in the 5-strand group before preparation of the 5-strand graft. The mean graft diameter of the 4-strand grafts was similar in both groups: 7.3 ± 0.3 mm in group A and 7.5 ± 0.8 mm in group B (P = .72). There was no statistically significant difference between the 2 groups of patients in terms of the Lysholm score, Knee Injury and Osteoarthritis Outcome Score (KOOS) Symptoms, KOOS Pain, KOOS Activities of Daily Living, KOOS Sports and KOOS Quality of Life scores. There were no postoperative complications of wound infection in both groups of patients. There was one case of graft rupture (4.8%) in the 4-strand group, which required revision reconstruction with patellar tendon graft 9 months postoperatively. There was no case of graft rupture in the 5-strand group (P = .29). Conclusions: The 5-strand hamstring graft technique provides a graft with significantly larger graft diameter than the quadrupled graft technique, with satisfactory short- to medium-term outcomes. The 5-strand graft is therefore a useful technique to increase the graft size when faced with the problem of small hamstring graft. Level of Evidence: Level II, prospective cohort study.

Unexpected deaths after endocrine surgery: learning from rare events using a national audit of surgical mortality.

BACKGROUND: The mortality rate is low in endocrine surgery, making it a difficult outcome to use for quality improvement in individual units. Lessons from population data sets are of value in improving outcomes. Data from the Australian and New Zealand Audit of Surgical Mortality (ANZASM) were used here to understand and elucidate potential systems issues that may contribute to preventable deaths. METHODS: ANZASM data relating to 30-day mortality after thyroidectomy, parathyroidectomy, and adrenalectomy from 2009 to 2020 were reviewed. Mortality rates were calculated using billing data. Thematic analysis of independent assessor reports was conducted to produce a coding framework. RESULTS: A total of 67 deaths were reported, with an estimated mortality rate of 0.03-0.07 per cent (38 for thyroidectomy (0.03-0.06 per cent), 16 for parathyroidectomy (0.03-0.06 per cent), 13 for adrenalectomy (0.15-0.33 per cent)). Twenty-seven deaths (40 per cent) were precipitated by clinically significant adverse events, and 18 (27 per cent) were judged to be preventable by independent ANZASM assessors. Recurrent themes included inadequate preoperative assessment, lack of anticipation of intraoperative pitfalls, and failure to recognize and effectively address postoperative complications. Several novel themes were reiterated, such as occult ischaemic heart disease associated with death after parathyroid surgery, unexpected intraoperative difficulties from adrenal metastasis, and complications due to anticoagulation therapy after thyroid surgery. CONCLUSION: This study represents a large-scale national report of deaths after endocrine surgery and provides insights into these rare events. Although the overall mortality rate is low, 27 per cent of deaths involved systems issues that were preventable following independent peer review.

Reconstitution of surface lipoprotein translocation through the Slam translocon.

Surface lipoproteins (SLPs) are peripherally attached to the outer leaflet of the outer membrane in many Gram-negative bacteria, playing significant roles in nutrient acquisition and immune evasion in the host. While the factors that are involved in the synthesis and delivery of SLPs in the inner membrane are well characterized, the molecular machinery required for the movement of SLPs to the surface are still not fully elucidated. In this study, we investigated the translocation of a SLP TbpB through a Slam1-dependent pathway. Using purified components, we developed an in vitro translocation assay where unfolded TbpB is transported through Slam1-containing proteoliposomes, confirming Slam1 as an outer membrane translocon. While looking to identify factors to increase translocation efficiency, we discovered the periplasmic chaperone Skp interacted with TbpB in the periplasm of Escherichia coli. The presence of Skp was found to increase the translocation efficiency of TbpB in the reconstituted translocation assays. A knockout of Skp in Neisseria meningitidis revealed that Skp is essential for functional translocation of TbpB to the bacterial surface. Taken together, we propose a pathway for surface destined lipoproteins, where Skp acts as a holdase for Slam-mediated TbpB translocation across the outer membrane.

Actinobacillus utilizes a binding protein-dependent ABC transporter to acquire the active form of vitamin B6.

Bacteria require high-efficiency uptake systems to survive and proliferate in nutrient-limiting environments, such as those found in host organisms. ABC transporters in the bacterial plasma membrane provide a mechanism for transport of many substrates. In this study, we examine an operon containing a periplasmic binding protein in Actinobacillus for its potential role in nutrient acquisition. The electron density map of 1.76 Å resolution obtained from the crystal structure of the periplasmic binding protein was best fit with a molecular model containing a pyridoxal-5'-phosphate (P5P/pyridoxal phosphate/the active form of vitamin B6) ligand within the protein's binding site. The identity of the P5P bound to this periplasmic binding protein was verified by isothermal titration calorimetry, microscale thermophoresis, and mass spectrometry, leading us to name the protein P5PA and the operon P5PAB. To illustrate the functional utility of this uptake system, we introduced the P5PAB operon from Actinobacillus pleuropneumoniae into an Escherichia coli K-12 strain that was devoid of a key enzyme required for P5P synthesis. The growth of this strain at low levels of P5P supports the functional role of this operon in P5P uptake. This is the first report of a dedicated P5P bacterial uptake system, but through bioinformatics, we discovered homologs mainly within pathogenic representatives of the Pasteurellaceae family, suggesting that this operon exists more widely outside the Actinobacillus genus.

Speaking Up and Speaking Out: Gender Diversity in the Scientific Programme of the Royal Australasian College of Surgeons Annual Scientific Congress.

BACKGROUND: Disparities in gender representation at medical meetings have been documented despite women representing half of medical school graduating classes. Lack of role models is touted as one of a myriad of factors that perpetuate gender imbalance, particularly in the field of surgery. We evaluated the trend in gender distribution of participants at the Royal Australasian College of Surgeons (RACS) Annual Scientific Congress (ASC) and whether there was a correlation between the gender distribution of the organising committee and speakers and chairpersons invited to attend. METHODS: RACS ASC programmes from 2013 to 2018 were retrospectively analysed, examining the gender distribution of speakers, chairpersons and conveners. Trend analysis of distribution was performed, and a generalized linear mixed model was used to investigate the effect of the gender of the conveners on gender of session chairpersons and speakers. RESULTS: Between 2013 and 2018, there were non-significant increases in female speakers invited to speak from 14.9 to 21.7% (p = 0.064) and female conveners appointed from 11 to 19% (p = 0.115), but there was a significant increase in female chairs from 9.6 to 21.6% p < 0.001). Female conveners were 3 times more likely to invite female speakers than male conveners (p < 0.001) and were 20 times more likely to invite female chairs than male conveners (p < 0.001). CONCLUSION: Visible role models are important in the pursuit of gender equity in surgery in order to break down stereotypes and the hidden curriculum. Intentional effort is required to achieve parity, and such efforts could include appointing more women to organising committees of scientific meetings.

Gender Representation by Specialty Track at Surgical Meetings: The American and Australasian Experiences.

BACKGROUND: We compared the representation of women panelists at two large, general interest surgical meetings: the American College of Surgeons (ACS) Clinical Congress and Royal Australasian College of Surgeons (RACS) Scientific Congress. MATERIALS AND METHODS: We performed comprehensive analyses of panels and panelists at ACS and RACS meetings (2013-2018). Manual review was conducted to determine counts and proportions of invited panelists by gender. We made within- and between-meeting comparisons regarding gender representation by specialty track. Tracks were characterized after our review of meeting programs. RESULTS: There were 4542 panelists and 1390 panels at RACS from 2013 to 2018. At ACS, there were 3363 panelists over 693 panels. The specialty tracks with the highest proportion of men-only panels were transplant (75%) and cardiothoracic (63%) at ACS and cardiothoracic (83%) and multidisciplinary (81%) at RACS. The lowest proportions of men-only panels were in breast and pediatric surgery at ACS (5% and 11%, respectively) and breast and rural surgery at RACS (24% and 36%, respectively). At ACS, the highest proportions of women panelists were on panels in breast (63%) and endocrine surgery (48%) and in breast (44%) and rural surgery (33%) at RACS, while the lowest proportion of women panelists were in transplant (10%) and cardiothoracic (14%) at ACS and multidisciplinary (8%) and cardiothoracic (7%) at RACS. CONCLUSIONS: There is a persistent difference in gender representation at surgical meetings, particularly within certain subspecialties. Program chairs and committees could increase the proportion of women by focusing on who serves as panelists overall and within specialty tracks.

The scaffold-protein IQGAP1 enhances and spatially restricts the actin-nucleating activity of Diaphanous-related formin 1 (DIAPH1).

The actin cytoskeleton is a dynamic array of filaments that undergoes rapid remodeling to drive many cellular processes. An essential feature of filament remodeling is the spatio-temporal regulation of actin filament nucleation. One family of actin filament nucleators, the Diaphanous-related formins, is activated by the binding of small G-proteins such as RhoA. However, RhoA only partially activates formins, suggesting that additional factors are required to fully activate the formin. Here we identify one such factor, IQ motif containing GTPase activating protein-1 (IQGAP1), which enhances RhoA-mediated activation of the Diaphanous-related formin (DIAPH1) and targets DIAPH1 to the plasma membrane. We find that the inhibitory intramolecular interaction within DIAPH1 is disrupted by the sequential binding of RhoA and IQGAP1. Binding of RhoA and IQGAP1 robustly stimulates DIAPH1-mediated actin filament nucleation in vitro In contrast, the actin capping protein Flightless-I, in conjunction with RhoA, only weakly stimulates DIAPH1 activity. IQGAP1, but not Flightless-I, is required to recruit DIAPH1 to the plasma membrane where actin filaments are generated. These results indicate that IQGAP1 enhances RhoA-mediated activation of DIAPH1 in vivo Collectively these data support a model where the combined action of RhoA and an enhancer ensures the spatio-temporal regulation of actin nucleation to stimulate robust and localized actin filament production in vivo.

Tweeting the meeting: an analysis of Twitter activity at the Royal Australasian College of Surgeons Annual Scientific Congress from 2015 to 2018.

BACKGROUND: Twitter has been shown to expand the audience and impact of material discussed at medical conferences, however, this phenomenon has not been analysed at the Royal Australasian College of Surgeons Annual Scientific Congress. The purpose of this study is to document the amount of Twitter activity at the Annual Scientific Congress, and to describe the way delegates use Twitter. METHODS: The number of tweets, retweets and contributors from the 2015 to 2018 congresses were determined using the Twitter advanced search function. Tweets were categorized broadly as academic, social or promotional. Union Metrics, an online software, was used to calculate estimates of impact including impressions and reach at the 2018 congress. Popular topics of discussion at all congresses were defined and counted. RESULTS: Twelve thousand five hundred and eight-six tweets were created with the official hashtag at the time of the four congresses. Activity increased over time except in the number of original tweets between the 2016 and 2018 congresses. Sixty-six percent of the tweets were directly related to congress content, and 23% were social in nature. At the 2018 congress, 16-34% of contributors were matched with a congress delegate. CONCLUSION: The tweets analysed were mainly informative. Twitter expanded the audience for material discussed at the Annual Scientific Congresses, and the amount of Twitter activity generally increased. It is in the interest of conference organizers to encourage and regulate Twitter use for maximum effectiveness because it is a powerful tool and use is likely to continue increasing in future.

Trends in Gender Representation at the American College of Surgeons Clinical Congress and the Academic Surgical Congress: A Mixed Picture of Progress.

BACKGROUND: There has been increasing attention to gender inequity in speakers at professional meetings. The aim of this study was to evaluate temporal trends in representation of women at the Academic Surgical Congress (ASC) and American College of Surgeons Clinical Congress (CC), 2 prominent general interest, national surgical meetings. STUDY DESIGN: We reviewed ASC (2014-2019) and CC (2013-2018) meeting programs to determine counts and proportions of invited panelists and moderators by gender, including the frequency of men-only panels. We conducted trend analyses to assess for temporal change in gender representation and univariate tests of association between different measures of gender representation. RESULTS: The overall proportions of women panelists were 35% (ASC) and 28% (CC). There was a significant increase in the proportion of women panelists over the study period at the CC (23% to 34%, p = 0.007) but not at the ASC (37% to 36%, p = 0.79). The proportion of men-only panels decreased significantly over time at the CC (38% to 23%, p = 0.04), but not at the ASC (23% to 17%, p = 0.50), while the proportion of moderators at the ASC increased significantly (31% to 43%, p = 0.01), but not at the CC (29% to 37%, p = 0.40). CONCLUSIONS: Women remain in the minority of panelists and moderators at the ASC and CC meetings, and approximately 1 in 5 panels are composed entirely of men. Although progress has been made at both meetings, ongoing and deliberate attention is needed to ensure continued progress toward the goal of equitable gender representation in academic surgery.

Global landscape of cell envelope protein complexes in Escherichia coli.

Bacterial cell envelope protein (CEP) complexes mediate a range of processes, including membrane assembly, antibiotic resistance and metabolic coordination. However, only limited characterization of relevant macromolecules has been reported to date. Here we present a proteomic survey of 1,347 CEPs encompassing 90% inner- and outer-membrane and periplasmic proteins of Escherichia coli. After extraction with non-denaturing detergents, we affinity-purified 785 endogenously tagged CEPs and identified stably associated polypeptides by precision mass spectrometry. The resulting high-quality physical interaction network, comprising 77% of targeted CEPs, revealed many previously uncharacterized heteromeric complexes. We found that the secretion of autotransporters requires translocation and the assembly module TamB to nucleate proper folding from periplasm to cell surface through a cooperative mechanism involving the ß-barrel assembly machinery. We also establish that an ABC transporter of unknown function, YadH, together with the Mla system preserves outer membrane lipid asymmetry. This E. coli CEP 'interactome' provides insights into the functional landscape governing CE systems essential to bacterial growth, metabolism and drug resistance.

Identification of a Large Family of Slam-Dependent Surface Lipoproteins in Gram-Negative Bacteria.

The surfaces of many Gram-negative bacteria are decorated with soluble proteins anchored to the outer membrane via an acylated N-terminus; these proteins are referred to as surface lipoproteins or SLPs. In Neisseria meningitidis, SLPs such as transferrin-binding protein B (TbpB) and factor-H binding protein (fHbp) are essential for host colonization and infection because of their essential roles in iron acquisition and immune evasion, respectively. Recently, we identified a family of outer membrane proteins called Slam (Surface lipoprotein assembly modulator) that are essential for surface display of neisserial SLPs. In the present study, we performed a bioinformatics analysis to identify 832 Slam related sequences in 638 Gram-negative bacterial species. The list included several known human pathogens, many of which were not previously reported to possess SLPs. Hypothesizing that genes encoding SLP substrates of Slams may be present in the same gene cluster as the Slam genes, we manually curated neighboring genes for 353 putative Slam homologs. From our analysis, we found that 185 (~52%) of the 353 putative Slam homologs are located adjacent to genes that encode a protein with an N-terminal lipobox motif. This list included genes encoding previously reported SLPs in Haemophilus influenzae and Moraxella catarrhalis, for which we were able to show that the neighboring Slams are necessary and sufficient to display these lipoproteins on the surface of Escherichia coli. To further verify the authenticity of the list of predicted SLPs, we tested the surface display of one such Slam-adjacent protein from Pasteurella multocida, a zoonotic pathogen. A robust Slam-dependent display of the P. multocida protein was observed in the E. coli translocation assay indicating that the protein is a Slam-dependent SLP. Based on multiple sequence alignments and domain annotations, we found that an eight-stranded beta-barrel domain is common to all the predicted Slam-dependent SLPs. These findings suggest that SLPs with a TbpB-like fold are found widely in Proteobacteria where they exist with their interaction partner Slam. In the future, SLPs found in pathogenic bacteria can be investigated for their role in virulence and may also serve as candidates for vaccine development.

Lactoferrin binding protein B - a bi-functional bacterial receptor protein.

Lactoferrin binding protein B (LbpB) is a bi-lobed outer membrane-bound lipoprotein that comprises part of the lactoferrin (Lf) receptor complex in Neisseria meningitidis and other Gram-negative pathogens. Recent studies have demonstrated that LbpB plays a role in protecting the bacteria from cationic antimicrobial peptides due to large regions rich in anionic residues in the C-terminal lobe. Relative to its homolog, transferrin-binding protein B (TbpB), there currently is little evidence for its role in iron acquisition and relatively little structural and biophysical information on its interaction with Lf. In this study, a combination of crosslinking and deuterium exchange coupled to mass spectrometry, information-driven computational docking, bio-layer interferometry, and site-directed mutagenesis was used to probe LbpB:hLf complexes. The formation of a 1:1 complex of iron-loaded Lf and LbpB involves an interaction between the Lf C-lobe and LbpB N-lobe, comparable to TbpB, consistent with a potential role in iron acquisition. The Lf N-lobe is also capable of binding to negatively charged regions of the LbpB C-lobe and possibly other sites such that a variety of higher order complexes are formed. Our results are consistent with LbpB serving dual roles focused primarily on iron acquisition when exposed to limited levels of iron-loaded Lf on the mucosal surface and effectively binding apo Lf when exposed to high levels at sites of inflammation.

Disease recurrence and survival in patients with multifocal breast cancer: a follow-up study with 7-year results.

BACKGROUND: Few studies have shown that multifocal breast cancer (MBC) has poorer outcomes compared with unifocal breast cancer (UBC). Currently, there is no long-term data on disease recurrence and survival in patients with MBC. The aim of this study is to evaluate whether patients with MBC have worse outcomes compared with UBC in respect to disease recurrence and survival. METHODS: This is a retrospective study of patients diagnosed with stage I-III MBC from 2000 to 2007 in comparison with UBC with a median follow-up of 7 years. Prognostic factors were prospectively collected from the breast cancer unit database. Univariate and multivariable analyses for disease recurrence and survival were performed as well as Kaplan-Meier curves. RESULTS: A total of 152 patients were included; 75 with MBC, 77 with UBC. The multifocal group was treated more aggressively with mastectomy (73% versus 25%, P < 0.0001) and chemotherapy (53% versus 42%). Breast cancer recurred in nine (11.7%) patients in the UBC group and nine (12%) patients in the MBC group respectively (hazard ratio (HR): 1.13, 95% confidence interval (CI): 0.45-2.86, P = 0.794). There were 10 (13%) mortalities in the unifocal group as compared with 11 (14.7%) in the multifocal group (HR: 1.02, 95% CI: 0.42-2.48, P = 0.969). There were no statistically significant differences in the all-cause mortality and disease recurrence rates between both groups. DISCUSSION: There were no statistically significant differences in disease recurrence or mortality rates between MBC and UBC at a median follow-up of 7 years. However, patients in the MBC group received more aggressive treatment than the unifocal group.

'AND' logic gates at work: Crystal structure of Rad53 bound to Dbf4 and Cdc7.

Forkhead-associated (FHA) domains are phosphopeptide recognition modules found in many signaling proteins. The Saccharomyces cerevisiae protein kinase Rad53 is a key regulator of the DNA damage checkpoint and uses its two FHA domains to interact with multiple binding partners during the checkpoint response. One of these binding partners is the Dbf4-dependent kinase (DDK), a heterodimer composed of the Cdc7 kinase and its regulatory subunit Dbf4. Binding of Rad53 to DDK, through its N-terminal FHA (FHA1) domain, ultimately inhibits DDK kinase activity, thereby preventing firing of late origins. We have previously found that the FHA1 domain of Rad53 binds simultaneously to Dbf4 and a phosphoepitope, suggesting that this domain functions as an 'AND' logic gate. Here, we present the crystal structures of the FHA1 domain of Rad53 bound to Dbf4, in the presence and absence of a Cdc7 phosphorylated peptide. Our results reveal how the FHA1 uses a canonical binding interface to recognize the Cdc7 phosphopeptide and a non-canonical interface to bind Dbf4. Based on these data we propose a mechanism to explain how Rad53 enhances the specificity of FHA1-mediated transient interactions.

Slam is an outer membrane protein that is required for the surface display of lipidated virulence factors in Neisseria.

Lipoproteins decorate the surface of many Gram-negative bacterial pathogens, playing essential roles in immune evasion and nutrient acquisition. In Neisseria spp., the causative agents of gonorrhoea and meningococcal meningitis, surface lipoproteins (SLPs) are required for virulence and have been extensively studied as prime candidates for vaccine development. However, the machinery and mechanism that allow for the surface display of SLPs are not known. Here, we describe a transposon (Tn5)-based search for the proteins required to deliver SLPs to the surface of Neisseria meningitidis, revealing a family of proteins that we have named the surface lipoprotein assembly modulator (Slam). N. meningitidis contains two Slam proteins, each exhibiting distinct substrate preferences. The Slam proteins are sufficient to reconstitute SLP transport in laboratory strains of Escherichia coli, which are otherwise unable to efficiently display these lipoproteins on their cell surface. Immunoprecipitation and domain probing experiments suggest that the SLP, TbpB, interacts with Slam during the transit process; furthermore, the membrane domain of Slam is sufficient for selectivity and proper surface display of SLPs. Rather than being a Neisseria-specific factor, our bioinformatic analysis shows that Slam can be found throughout proteobacterial genomes, indicating a conserved but until now unrecognized virulence mechanism.

A method for measuring binding constants using unpurified in vivo biotinylated ligands.

Obtaining accurate kinetics and steady-state binding constants for biomolecular interactions normally requires pure and homogeneous protein preparations. Furthermore, in many cases, one of the ligands must be labeled. Over the past decade, several technologies have been introduced that allow for the measurement of kinetics constants for multiple different interactions in parallel. One such technology is bio-layer interferometry (BLI), which has been used to develop systems that can measure up to 96 biomolecular interactions simultaneously. However, despite the ever-increasing throughput of the tools available for measuring protein-protein interactions, the preparation of pure protein still remains a bottleneck in the process of producing high-quality kinetics data. Here, we show that high-quality binding data can be obtained using soluble lysate fractions containing protein that has been biotinylated in vivo using BirA and then applied to BLI sensors without further purification. Furthermore, we show that BirA ligase does not necessarily need to be co-overexpressed with the protein of interest for biotinylation of the biotin acceptor peptide to occur, suggesting that the activity of endogenous BirA in Escherichia coli is sufficient for producing enough biotinylated protein for a binding experiment.