100 ancient genomes show repeated population turnovers in Neolithic Denmark.

Major migration events in Holocene Eurasia have been characterized genetically at broad regional scales1-4. However, insights into the population dynamics in the contact zones are hampered by a lack of ancient genomic data sampled at high spatiotemporal resolution5-7. Here, to address this, we analysed shotgun-sequenced genomes from 100 skeletons spanning 7,300 years of the Mesolithic period, Neolithic period and Early Bronze Age in Denmark and integrated these with proxies for diet (13C and 15N content), mobility (87Sr/86Sr ratio) and vegetation cover (pollen). We observe that Danish Mesolithic individuals of the Maglemose, Kongemose and Ertebølle cultures form a distinct genetic cluster related to other Western European hunter-gatherers. Despite shifts in material culture they displayed genetic homogeneity from around 10,500 to 5,900 calibrated years before present, when Neolithic farmers with Anatolian-derived ancestry arrived. Although the Neolithic transition was delayed by more than a millennium relative to Central Europe, it was very abrupt and resulted in a population turnover with limited genetic contribution from local hunter-gatherers. The succeeding Neolithic population, associated with the Funnel Beaker culture, persisted for only about 1,000 years before immigrants with eastern Steppe-derived ancestry arrived. This second and equally rapid population replacement gave rise to the Single Grave culture with an ancestry profile more similar to present-day Danes. In our multiproxy dataset, these major demographic events are manifested as parallel shifts in genotype, phenotype, diet and land use.

Cyclic-di-GMP regulation promotes survival of a slow-replicating subpopulation of intracellular Salmonella Typhimurium.

Salmonella Typhimurium can invade and survive within macrophages where the bacterium encounters a range of host environmental conditions. Like many bacteria, S. Typhimurium rapidly responds to changing environments by the use of second messengers such as cyclic di-GMP (c-di-GMP). Here, we generate a fluorescent biosensor to measure c-di-GMP concentrations in thousands of individual bacteria during macrophage infection and to define the sensor enzymes important to c-di-GMP regulation. Three sensor phosphodiesterases were identified as critical to maintaining low c-di-GMP concentrations generated after initial phagocytosis by macrophages. Maintenance of low c-di-GMP concentrations by these phosphodiesterases was required to promote survival within macrophages and virulence for mice. Attenuation of S Typhimurium virulence was due to overproduction of c-di-GMP-regulated cellulose, as deletion of the cellulose synthase machinery restored virulence to a strain lacking enzymatic activity of the three phosphodiesterases. We further identified that the cellulose-mediated reduction in survival was constrained to a slow-replicating persister population of S. Typhimurium induced within the macrophage intracellular environment. As utilization of glucose has been shown to be required for S. Typhimurium macrophage survival, one possible hypothesis is that this persister population requires the glucose redirected to the synthesis of cellulose to maintain a slow-replicating, metabolically active state.

The dual influence of the reed resonance frequency and tonehole lattice cutoff frequency on sound production and radiation of a clarinet-like instrument.

The internal and external spectra of woodwind reed instruments are partially determined by the tonehole lattice cutoff and reed resonance frequencies. Because they can impact the spectrum in similar ways, a study of one without accounting for the other risks incomplete or false conclusions. Here, the dual effects of the cutoff and reed resonance frequencies are investigated using digital synthesis with clarinet-like academic resonators. It is shown that the odd and even harmonics have similar amplitudes at and above the cutoff frequency or reed resonance frequency, whichever is lowest. However, because the resonators radiate efficiently at the cutoff, it has the additional role of reinforcing the amplitude of both the odd and even harmonics in the external spectrum. The spectra are analyzed using the single value descriptors playing frequency, spectral centroid (SC), odd/even ratio (OER), and brightness as a function of the musician mouth pressure. Higher reed resonances correspond to higher values for all descriptors. The OER and brightness increase with resonator cutoff frequency, whereas the SC exhibits more complicated trends. The reed resonance has a larger impact on the "playing condition oscillation threshold," implying that it may have a more important role in sustaining auto-oscillation.

The bassoon tonehole lattice: Links between the open and closed holes and the radiated sound spectrum.

The acoustics of the bassoon has been the subject of relatively few studies compared with other woodwind instruments. One reason for this may lie in its complicated resonator geometry, which includes irregularly spaced toneholes with chimney heights ranging from 3 to 31 mm. The current article evaluates the effect of the open and closed tonehole lattice (THL) on the acoustic response of the bassoon resonator. It is shown that this response can be divided into three distinct frequency bands that are determined by the open and closed THL: below 500 Hz, 500-2200 Hz, and above 2200 Hz. The first is caused by the stopband of the open THL, where the low frequency effective length of the instrument is determined by the location of the first open tonehole. The second is due to the passband of the open THL, such that the modes are proportional to the total length of the resonator. The third is due to the closed THL, where part of the acoustical power is trapped within the resonator. It is proposed that these three frequency bands impact the radiated spectrum by introducing a formant in the vicinity of 500 Hz and suppressing radiation above 2200 Hz for most first register fingerings.

The cellular microbiology of Salmonellae interactions with macrophages.

Salmonellae are important enteric pathogens that cause gastroenteritis and systemic illnesses. Macrophages are important components of both the innate and acquired immune system, acting as phagocytes with significant antimicrobial killing activities that present antigen to the adaptive immune system. Macrophages can also be cultured from a variety of sites as primary cells, and the study of the survival and interactions of Salmonellae with these cells is a very early model of infection and cellular microbiology. This review traces the history of discoveries made using Salmonellae infection of macrophages and addresses the possibility of future research in this area, in particular with regards to understanding the complexity of individual bacteria and macrophage cell variability and how such heterogeneity may alter the outcome of infection.

Superficial Basal Cell Cancers Demonstrate Higher Rates of Mixed Histology on High-Risk Anatomical Sites.

BACKGROUND: The Mohs Appropriate Use Criteria (MAUC) have come into question recently regarding the most appropriate treatment for superficial basal cell carcinoma (sBCC). At the heart of this debate is the limited body of evidence describing tumor behavior of sBCC based on clinical factors relevant to the MAUC. OBJECTIVE: To determine whether sBCC is more likely to harbor aggressive subtypes in high-risk anatomical locations and in immunocompromised patients. MATERIALS AND METHODS: A single institution retrospective review produced 133 evaluable Mohs cases performed on sBCC over a 10-year period. All slides from the respective cases were reviewed for the presence of histologic patterns other than known sBCC. Cases were then grouped by both MAUC anatomical zone (H, M, and L) and patient immune status for statistical analysis. RESULTS: A significantly higher rate of mixed histology (MH) was observed when comparing Zone H with Zone L across all patients, healthy patients, and immunocompromised patients. The same was true when comparing Zone M with Zone L for all patients and healthy patients (immunocompromised did not reach significance). CONCLUSION: The authors' data very clearly demonstrate a higher rate of MH in sBCC of the head and neck which provides strong support to the current MAUC scoring.

An evolving presentation of cutaneous tuberculosis.

Tuberculosis is a fairly common disease in the United States and around the world, newly infecting ten million people throughout the world per year. Despite the pervasiveness of tuberculosis, cutaneous tuberculosis (CTB) rarely manifests worldwide. Tuberculous infections of the skin arise in several distinct variants that can be classified as either multibacillary or paucibacillary; each subtype within these categories presents with its own morphological and histological findings. The diagnosis of CTB can prove clinically challenging as its variants mimic many conditions dermatologist encounter on a daily basis. Additionally, tissue confirmation is difficult. We report a case of CTB which evolved from a lupus vulgaris presentation to the metastatic tuberculous abscess variant.

Review of systemic agents in the treatment of advanced cutaneous squamous cell carcinoma.

Advanced cutaneous squamous cell carcinoma (cSCC) accounts for only 5% of all cases of cSCC but up to 60% of disease related deaths. Historically, this disease has lacked effective treatment options due to a combination of poor response rate, poor response durability and significant treatment-associated morbidity. Autumn of 2018 marked the first time ever that an agent received US FDA approval for advanced cSCC and the future is looking much brighter for this previously neglected patient population. The purpose of this article is to review the various systemic treatment options for advanced cSCC moving from the past to the present, highlighting their relative merits and shortcomings, and to briefly speculate on future developments in the field of advanced cSCC.

Complications associated with intimate body piercings.

Intimate body piercings involving the nipple and genitalia have increased in prevalence in both men and women. Despite this increase, there is a deficiency in the literature regarding the short and long-term complications of body piercings, including an increased risk of infection, malignancy, and structural damage to the associated tissue. Breast abscesses associated with nipple piercing can be mistaken as inflammatory carcinoma. Male genital piercings have been associated with urethral rupture, paraphimosis, urethral obstruction, scar formation, and squamous cell carcinoma, whereas female genital piercings may lead to a higher risk of pregnancy and sexually transmitted infections. There are additional problems related to piercings during pregnancy and thereafter. Nipple piercings can hinder breast feeding by inhibiting the milk letdown reflex, increasing nipple sensitivity, and causing discomfort to the infant. Removal of genital piercings during pregnancy could introduce bacteria into the piercing tract, but retaining the piercings could theoretically hinder childbirth. Prevention of complications is critical. Patients must understand the risks of piercings and disclose relevant medical conditions to the practitioner before the procedure. The piercings should be carried out in a hygienic and sterile manner. Finally, physicians should maintain a non-judgmental attitude to encourage patients to seek medical care for complications.

No pain no gain: tender nodules in a competitive bodybuilder.

The subculture of bodybuilding is rife with people willing to do whatever is necessary to achieve the perfect physique. One particularly concerning behavior is the injection of site-enhancing-oils (SEO) into lagging muscle groups to achieve instant size and symmetry. The typical SEO is a combination of lidocaine, alcohol, and oil; it is rarely, if ever, administered by a qualified professional. As a result, there are a variety of potential complications that can manifest in the skin and other organ systems. In our case, a 41-year-old former competitive bodybuilder was referred to our clinic for excision of a subcutaneous nodule. The initial histopathology was concerning for lymphoma, but a more thorough history and review of systems were completely negative. The patient underwent a negative systemic lymphoma workup and it was not until we discussed the prospects of radiation and other forms of treatment that he revealed a history of SEO use, as well as other identical nodules on his body. Subsequent excisions revealed a more classic sclerosing lipogranuloma-type reaction pattern. Owing to the taboo nature of SEOs, most patients are reluctant to provide this vital piece of historical information, highlighting the importance of patient rapport and clinical-pathologic correlation in our specialty.

Corrigendum: Complications associated with intimate body piercings.

The original article was published on July15, 2017 and corrected on August 15, 2018.The revised version of the article includes a correction to the spelling of an author. The change appears in the revised online PDF copy of this article.

A case of radiation-induced bullous morphea/lichen sclerosus overlap in a breast cancer patient.

Radiation induced morphea (RIM) is an increasingly common complication of radiation treatment for malignancy as early detection has made more patients eligible for non-surgical treatment options. In many cases, the radiation oncologist is the first person to learn of the initial skin changes, often months before a dermatologist sees them. In this paper we present a breast cancer patient who developed a rare bullous variant of RIM, which delayed her diagnosis and subsequent treatment. It is imperative to diagnose RIM early as it carries significant morbidity and permanent deformity if left untreated. The lesions typically present within 1 year of radiation therapy and extend beyond the radiated field. RIM is often mistaken for radiation dermatitis or cellulitis. Bullae, when present, are often hemorrhagic in appearance, which can serve as another clinical clue. It is important to refer these patients for a full gynecologic exam as there can be concurrent anogenital lichen sclerosus et atrophicus which is both debilitating and carries a long term risk for squamous cell carcinoma. Treatment with systemic agents is often necessary, and can be managed by a dermatologist. The most proven regimen in the literature appears to be methotrexate, with our without concurrent narrow band UVB phototherapy.

Differences in gut bacterial community composition between modern and slower-growing broiler breeder lines: Implications of growth selection on microbiome composition.

In the last century broiler chicken lines have undergone an extensive breeding regime aimed primarily at growth and high meat yield. It is not known if breeding has also resulted in a change to the broiler breeder's associated gut microbiota. Here we compared the gut microbiota of 37-week-old commercial Cobb breeding dams with dams from a broiler Legacy line which has not undergone selection since 1986. The dams from both lines were kept together in the same shed under the same management protocol from day of hatch to avoid additional confounders. We chose this age to allow significant bacterial exchange, thus avoiding exposure dependent artifacts and so that we could compare dams at the same developmental state of adulthood and peak laying performance. Significant differences in the composition of the cecum bacterial communities were found. Bacteria of the genus Akkermansia, implicated in mucin degradation and associated with host metabolic health, accounted for 4.98% ± 5.04% of the Cobb cecum community, but were mostly absent from the ceca of the Legacy line dams. Inversely, Legacy dams had higher levels of Clostridiales, Lactobacillales and Aeromonadales. These results show that breeding has resulted in a change in the gut microbiota composition, likely by changing the physiological conditions in the mucosa. It remains unclear if changes in gut microbiota composition are a part of the mechanism affecting growth or are a secondary result of other physiological changes accelerating growth. Therefore, the identification of these changes opens the door to further targeted research.

Quantification of Microbial Fluorescent Sensors During Live Intracellular Infections.

The interaction of pathogens with their eukaryotic hosts during intracellular growth is critical to many diseases. However, the relative scarcity of pathogen biomolecules versus the abundant host biomolecule concentration can make quantitative evaluation of pathogen intracellular responses difficult. Recent years have seen an explosion in utilization of fluorescent proteins to serve as transcriptional reporters and biosensors for quantification of pathogen responses. Here, we describe a method to establish a fluorescent assay quantifying pathogen behavior during intracellular infection and to quantify these results at a single cell level. The sensitivity of these fluorescent assays permits the live observation of changing pathogen responses, while the ability to measure at a single cell level uncovers subpopulations of pathogens whose existence may be missed during the population-level assays often required to accumulate sufficient pathogen biomolecules for analysis.

Brucella melitensis cyclic di-GMP phosphodiesterase BpdA controls expression of flagellar genes.

Brucella melitensis encounters a variety of conditions and stimuli during its life cycle--including environmental growth, intracellular infection, and extracellular dissemination--which necessitates flexibility of bacterial signaling to promote virulence. Cyclic-di-GMP is a bacterial secondary signaling molecule that plays an important role in adaptation to changing environments and altering virulence in a number of bacteria. To investigate the role of cyclic-di-GMP in B. melitensis, all 11 predicted cyclic-di-GMP-metabolizing proteins were separately deleted and the effect on virulence was determined. Three of these cyclic-di-GMP-metabolizing proteins were found to alter virulence. Deletion of the bpdA and bpdB genes resulted in attenuation of virulence of the bacterium, while deletion of the cgsB gene produced a hypervirulent strain. In a Vibrio reporter system to monitor apparent alteration in levels of cyclic-di-GMP, both BpdA and BpdB displayed a phenotype consistent with cyclic-di-GMP-specific phosphodiesterases, while CgsB displayed a cyclic-di-GMP synthase phenotype. Further analysis found that deletion of bpdA resulted in a dramatic decrease in flagellar promoter activities, and a flagellar mutant showed similar phenotypes to the bpdA and bpdB mutant strains in mouse models of infection. These data indicate a potential role for regulation of flagella in Brucella melitensis via cyclic-di-GMP.

Toxin Glycan Binding: Lectin Keys Unlocking Host and Tissue Specificity.

In this issue of Cell Host and Microbe, Lee et al. define the glycan binding specificity of a variant of typhoid toxin produced by a non-typhoidal Salmonellae serotype. The authors elegantly demonstrate that tissue and host specificity of the toxin are related to specific glycan binding characteristics of the toxin.

Structure of an Inner Membrane Protein Required for PhoPQ-Regulated Increases in Outer Membrane Cardiolipin.

The Salmonella enterica subsp. enterica serovar Typhimurium PhoPQ two-component system is activated within the intracellular phagosome environment, where it promotes remodeling of the outer membrane and resistance to innate immune antimicrobial peptides. Maintenance of the PhoPQ-regulated outer membrane barrier requires PbgA, an inner membrane protein with a transmembrane domain essential for growth, and a periplasmic domain required for PhoPQ-activated increases in outer membrane cardiolipin. Here, we report the crystal structure of cardiolipin-bound PbgA, adopting a novel transmembrane fold that features a cardiolipin binding site in close proximity to a long and deep cleft spanning the lipid bilayer. The end of the cleft extends into the periplasmic domain of the protein, which is structurally coupled to the transmembrane domain via a functionally critical C-terminal helix. In conjunction with a conserved putative catalytic dyad situated at the middle of the cleft, our structural and mutational analyses suggest that PbgA is a multifunction membrane protein that mediates cardiolipin transport, a function essential for growth, and perhaps catalysis of an unknown enzymatic reaction.IMPORTANCE Gram-negative bacteria cause many types of infections and have become increasingly resistant to available antibiotic drugs. The outer membrane serves as an important barrier that protects bacteria against antibiotics and other toxic compounds. This outer membrane barrier function is regulated when bacteria are in host environments, and the protein PbgA contributes significantly to this increased barrier function by transporting cardiolipin to the outer membrane. We determined the crystal structure of PbgA in complex with cardiolipin and propose a model for its function. Knowledge of the mechanisms of outer membrane assembly and integrity can greatly contribute to the development of new and effective antibiotics, and this structural information may be useful in this regard.

Cemiplimab-rwlc as first and only treatment for advanced cutaneous squamous cell carcinoma.

Introduction: In September of 2018, the United States Federal Drug Administration (FDA) approved cemiplimab-rwlc (Libtayo) for advanced cutaneous squamous cell carcinoma (CSCC). Cemiplimab is an intravenous human monoclonal antibody directed against programmed cell death-1 receptor (PD-1). Cemiplimab blocks T-cell inactivation and enhances the immune system's anti-tumor response. Areas Covered: We review CSCC and the studies leading to cemiplimab's approval, including common side effects and safety issues experienced during the clinical trials. Expert Opinion: Immunotherapy, specifically checkpoint inhibitors, represents an increasingly utilized class of medications that is proving to be an effective treatment option for those with certain cancers. Over time, immunotherapy is likely to be the standard of care for immune-sensitive tumors. There are many challenges that the field faces, including the identification of reliable biomarkers to better predict response, decreasing toxicity, and the potential treatment of organ transplant patients.

Salmonella Translocated Effectors Recruit OSBP1 to the Phagosome to Promote Vacuolar Membrane Integrity.

Intracellular Salmonella use a type III secretion system (TTSS) to translocate effector proteins across the phagosome membrane and thus promote vacuole membrane tubulation, resulting in intracellular survival. This work demonstrates that the effector SseJ binds the eukaryotic lipid transporter oxysterol binding protein 1 (OSBP1). SseJ directs OSBP1 to the endosomal compartment in a manner dependent on the TTSS located on Salmonella pathogenicity island 2 (SPI2). OSBP1 localization is mediated by both SseJ and another OSBP1-binding SPI2 translocated effector, the deubiquitinase SseL. Deletion of both SseJ and SseL reduced vacuolar integrity with increased bacteria released into the eukaryotic cytoplasm of epithelial cells, indicating that their combined activities are necessary for vacuole membrane stability. Cells knocked down for OSBP1 or deleted for the OSBP1-binding proteins VAPA/B also demonstrate loss of vacuole integrity, consistent with the hypothesis that OSBP1 recruitment is required for SPI2-mediated alterations that promote vacuolar integrity of salmonellae.

Genomic island 2 of Brucella melitensis is a major virulence determinant: functional analyses of genomic islands.

Brucella genomic islands (GIs) share similarities in their genomic organization to pathogenicity islands from other bacteria and are likely acquired by lateral gene transfer. Here, we report the identification of a GI that is important for the pathogenicity of Brucella melitensis. The deletion of GI-1, GI-5, or GI-6 did not affect bacterial growth in macrophages as well as their virulence in interferon regulatory factor 1-deficient (IRF-1(-/-)) mice, suggesting that these islands do not contribute to Brucella virulence. However, the deletion of GI-2 resulted in the attenuation of bacterial growth in macrophages and virulence in IRF-1(-/-) mice. The GI-2 mutant also displayed a rough lipopolysaccharide (LPS) phenotype indicated by acriflavin agglutination, suggesting that in vitro and in vivo attenuation is a result of LPS alteration. Further, systematic analysis of the entire GI-2 revealed two open reading frames (ORFs), BMEI0997 and I0998, that encode hypothetical sugar transferases and contribute to LPS alteration, as the deletion of either of these ORFs resulted in a rough phenotype similar to that of the GI-2 mutant. Complementation analyses indicated that in addition to I0997 and I0998, I0999 is required to restore the smooth LPS in the GI-2 mutant as well as its full in vitro and in vivo virulence. The I0999 sequence analysis suggested that it might function as a transporter to help facilitate the transport or linking of the O antigen to the LPS. Our study also indicated that the rough LPS resulting from the GI-2 deletion may affect pathogen-associated molecular pattern recognition by Toll-like receptors.