Geroscience: Aging and Oral Health Research.

Research in aging has significantly advanced; scientists are now able to identify interventions that slow the biologic aging processes (i.e., the "hallmarks of aging"), thus delaying the onset and progression of multiple diseases, including oral conditions. Presentations given during the 3-part session "Geroscience: Aging and Oral Health Research," held during the 2023 American Association for Dental, Oral, and Craniofacial Research meeting, are summarized in this publication. Speakers' topics spanned the translational research spectrum. Session 1 provided an overview of the geroscience and health span (disease-free and functional health throughout life) concepts. The common molecular mechanisms between oral cancer and aging were discussed, and research was presented that showed periodontal microflora as a potential factor in Alzheimer's disease progression. Session 2 focused on behavioral and social science aspects of aging and their oral health significance. The keynote provided evidence that loneliness and isolation can have major health effects. These social conditions, along with poor oral health, tooth loss, and cognitive decline, could potentially affect healthy eating ability and systemic health in older adults. Research could help elucidate the directions and pathways connecting these seemingly disparate conditions. Session 3 focused on the delivery of oral care in different settings and the many barriers to access care faced by older adults. Research is needed to identify and implement effective technology and strategies to improve access to dental care, including new delivery and financing mechanisms, workforce models, interprofessional provider education and practice, and use of big data from medical-dental integration of electronic health records. Research to improve the "oral health span," reduce oral health disparities, and increase health equity must be tackled at all levels from biologic pathways to social determinants of health and health policies.

Alzheimer PHF-tau aggregates do not spread tau pathology to the brain via the Retino-tectal projection after intraocular injection in male mouse models.

Neurofibrillary tangles (NFT), a neuronal lesion found in Alzheimer's disease (AD), are composed of fibrillary aggregates of modified forms of tau proteins. The propagation of NFT follows neuroanatomical pathways suggesting that synaptically connected neurons could transmit tau pathology by the recruitment of normal tau in a prion-like manner. Moreover, the intracerebral injection of pathological tau from AD brains induces the seeding of normal tau in mouse brain. Creutzfeldt-Jacob disease has been transmitted after ocular transplants of cornea or sclera and the scrapie agent can spread across the retino-tectal pathway after intraocular injection of scrapie mouse brain homogenates. In AD, a tau pathology has been detected in the retina. To investigate the potential risk of tau pathology transmission during eye surgery using AD tissue material, we have analysed the development of tau pathology in the visual pathway of mice models expressing murine tau, wild-type or mutant human tau after intraocular injection of pathological tau proteins from AD brains. Although these pathological tau proteins were internalized in retinal ganglion cells, they did not induce aggregation of endogenous tau nor propagation of a tau pathology in the retino-tectal pathway after a 6-month incubation period. These results suggest that retinal ganglion cells exhibit a resistance to develop a tau pathology, and that eye surgery is not a major iatrogenic risk of transmission of tau pathology, contrary to what has been observed for transmission of infectious prions in prion diseases.

Associations between Dentition Status and Nutritional Status in Community-Dwelling Older Adults.

BACKGROUND/OBJECTIVE: Older adults are at higher risk of malnutrition. The aim of this study was to explore associations between nutritional status and dentition status among older adults seeking care in a dental clinic. METHODS: This was a cross-sectional study of data from older adults (65-89 y) who received care at a northeastern US urban dental school clinic between June 2015 and June 2020 (N = 305). Clinical and demographic data were obtained from the electronic health record; nutritional status was determined using the Self-Mini Nutritional Assessment (Self-MNA), and odontograms and digital radiography were used to determine dental data. Adjusted multivariable models were used to explore associations between variables. RESULTS: The sample was 53.8% female with a median age of 72.0 y. The median Self-MNA score was 13, reflective of normal nutritional status; 29.5% were at risk of or had malnutrition. Median numbers of teeth and posterior and anterior occluding pairs of teeth (POP, AOP) were 18.0, 2.0, and 5.0, respectively. Those with normal nutritional status had significantly more teeth, POPs, and AOPs than those at risk of or with malnutrition (P = 0.015, P = 0.015, and P = 0.039, respectively). Every additional unit increase in the number of natural or restored teeth or POP was associated with significantly lower odds of being at risk of or with malnutrition (3% and 13%, respectively). Having functional dentition was associated with 46% lower odds of being at risk of or with malnutrition. CONCLUSION: This study demonstrated that older adults who had more teeth, better occlusion, and functional dentition were more likely to be of normal nutritional status than those who had less teeth, had poorer occlusion, and lacked functional dentition. Further research with larger, more diverse samples and varied measures of dentition are needed to better understand the associations between nutritional status and dentition status. KNOWLEDGE TRANSFER STATEMENT: The findings from this study suggest that older adults with fewer teeth and therefore less efficient occlusion are at higher risk for malnutrition than those with more teeth and better occlusion. Health care professionals should include screening for dentition and malnutrition as part of their routine practice to identify patients who may have tooth loss and be at risk of malnutrition and refer them accordingly for interventions to optimize oral health and nutritional status.

Tooth Loss and Nutritional Status in Older Adults: A Systematic Review and Meta-analysis.

BACKGROUND/OBJECTIVE: Older adults are at risk for tooth loss and compromised nutritional status. Our objective was to conduct a systematic review and meta-analysis to answer the following question: Among adults aged ≥60 y living in developed countries, what are the associations between tooth loss and nutritional status as assessed by a validated nutrition screening or assessment tool? METHODS: PRISMA guidelines were followed. PubMed, Scopus, CINAHL, Web of Science, and MEDLINE were searched for studies published in English between 2009 and 2019 that met inclusion criteria. Data extracted included study and participant characteristics, dentition, and nutritional status. Risk of bias was assessed with a modified Newcastle-Ottawa Scale. Random effects meta-analysis was used. RESULTS: Of the 588 unduplicated articles identified, 78 were reviewed in full text, and 7 met inclusion criteria. Six studies were combined for a meta-analysis, which revealed that individuals who were completely edentulous or who lacked functional dentition had a 21% increased likelihood of being at risk of malnutrition or being malnourished, as compared with those who were dentulous or had functionally adequate dentition (risk ratio, 1.21; 95% CI, 1.11 to 1.32; I2 = 70%). Whether the article statistically adjusted for medical history explained most of the heterogeneity in the pooled effect. CONCLUSIONS AND IMPLICATIONS: Findings suggest that older adults with tooth loss are at greater risk of malnutrition than those with functionally adequate dentition. Use of validated tools to assess risk of malnutrition in older adults with tooth loss is important to promote early intervention and referral to optimize nutrition and oral health status. Findings were limited by heterogeneity, risk of bias, and overall quality of the studies reviewed. Cohort studies that adjust for known confounders and use consistent approaches to assess tooth loss and nutritional status are needed. KNOWLEDGE TRANSFER STATEMENT: The results of this study suggest that older adults with tooth loss are at greater risk of malnutrition than those with functionally adequate dentition. Screening of this population for malnutrition by health care professionals, including dentists and dietitians, may result in corresponding referrals to optimize nutrition and oral health status. Further research is needed with consistent approaches to assess tooth loss and nutritional status.

A retrospective description of primary immuno-deficiency diseases at Red Cross War Memorial Children's Hospital, Cape Town, South Africa, 1975 - 2017.

BACKGROUND: The primary immunodeficiency diseases (PIDs) constitute a diverse and ever-expanding group of inborn errors affecting a wide range of immune functions. They are not well documented in sub-Saharan Africa. OBJECTIVES: To describe the spectrum of PIDs at a tertiary paediatric hospital. METHODS: A retrospective descriptive study of PIDs diagnosed at Red Cross War Memorial Children's Hospital, Cape Town, South Africa (SA), between 1975 and 2017 was undertaken. RESULTS: We identified 252 children with PIDs, spanning eight of the nine categories listed in the 2017 classification of the International Union of Immunological Societies. Predominantly antibody deficiencies, combined immunodeficiencies with associated syndromic features, and immunodeficiencies affecting cellular and humoral immunity accounted for most children with PIDs (n=199, 79.0%). The mean age (standard deviation) at diagnosis was 46 (50) months, and the male/female ratio was 1.5:1. There was a history of parental consanguinity in 3 cases (1.2%). Recurrent infection was the most prevalent presenting phenotype, manifesting in 177 patients (70.2%). Genetic or chromosomal confirmation was obtained in 42/252 cases (16.7%). Common interventions used to prevent infection were antimicrobial prophylaxis and immunoglobulin replacement therapy, administered to 95 (37.7%) and 93 (36.9%) of the patients, respectively. Six of 7 children who underwent haematopoietic stem cell transplantation (HSCT) had successful outcomes. The 7th patient died 2 months after HSCT from overwhelming infection. Although we could not account for the children lost to follow-up during the study period, 53 deaths were confirmed (21.0%). CONCLUSIONS: Several challenges exist in the recognition and treatment of children with PIDs in our setting. These include limited access to genetic diagnostics and HSCT. Suboptimal treatment options contribute to the overall mortality of PIDs in SA.

The GOLD Summit on chronic obstructive pulmonary disease in low- and middle-income countries.

Chronic obstructive pulmonary disease (COPD) is one of the top three causes of death worldwide, but governments and non-governmental organisations have not given its prevention and treatment the priority it requires. This is particularly true in low- and middle-income countries, where most of the people suffering from this disease live. The United Nations (UN) has targeted a reduction of premature deaths from non-communicable diseases (NCDs) by a third by 2030; however, a coordinated UN/World Health Organization (WHO) strategy to address the burden of COPD (one of the most important NCDs) is still lacking. To explore the extent of the problem and inform the development of policies to improve the situation, the Board of Directors of the Global Initiative for Chronic Obstructive Lung Disease (GOLD) held a 1-day Summit. The key themes that emerged were the need to ensure accurate data on prevalence, raise awareness of the disease among the public, healthcare professionals and governments, including the fact that COPD aetiology goes beyond smoking (and other inhaled pollutants) and includes poor lung development in early life, and ensure that spirometry and both pharmacological and non-pharmacological therapies are available and affordable. Here, we present the actions that must be taken to address the impact of COPD. We believe that the WHO is particularly well-positioned to co-ordinate an attack on COPD, and GOLD will do all it can to help and rally support.

The Eating Experience: Adaptive and Maladaptive Strategies of Older Adults with Tooth Loss.

OBJECTIVE: To explore the eating experience and eating-related quality of life (ERQOL) of community-dwelling older adults with tooth loss. METHOD: Nineteen older adults from the clinics of a northeast US dental school who met inclusion criteria (>65 y old, <20 teeth, and no dentures) composed the sample. For this mixed methods study, demographic characteristics, number and location of teeth, Mini Nutritional Assessment-Short Form score, and anthropometrics data were collected; semistructured interviews were conducted to collect in-depth information about the eating experience and ERQOL. Thematic analysis was completed with NVivo 12 software (QSR International). RESULTS: Participants' mean age was 71.3 y (SD = 5.2); 52.6% (n = 10) were women; 63.2% (n = 12) were Black or African American. The mean Mini Nutritional Assessment-Short Form score of 12.1 was reflective of normal nutrition status; 31.6% (n = 6) of patients were at risk for malnutrition or were malnourished. Fifteen percent (n = 3) were fully edentulous; 84.2% (n = 16) had 1 to 19 teeth (mean = 10.8, SD = 6.5). The 2 overarching themes identified were adaptive and maladaptive behavioral responses to tooth loss. Adaptive strategies included modification in food preparation and cooking methods, food texture selection, meal timing, and approaches to chewing. Maladaptive behaviors included food avoidance and limiting eating and smiling in front of others. Psychosocial factors, including finances, limited food choices and ERQOL, whereas the support of family and friends enhanced ERQOL according to participants. CONCLUSION: Older adults with tooth loss exhibit both adaptive and maladaptive behaviors that affect their eating experience, dietary intake, and ERQOL. While many expressed positive adaptive coping strategies, they also described maladaptive behaviors, including avoidance of healthy foods and limiting eating during social interactions, which may affect their nutritional status and overall health and well-being. Further research is needed to explore how duration and severity of tooth loss influence these behaviors and risk of malnutrition. Interprofessional approaches are needed to support positive adaptation and coping with tooth loss. KNOWLEDGE TRANSFER STATEMENT: The results of this study can be used by health professionals treating patients with tooth loss in an effort to improve their eating experience and eating-related quality of life. The findings provide data to support further studies and the need for evidence-based guidelines and educational materials to meet the unique needs of older adults with tooth loss.

Pluto's Interaction With Energetic Heliospheric Ions.

Pluto energies of a few kiloelectron volts and suprathermal ions with tens of kiloelectron volts and above. We measure this population using the Pluto Energetic Particle Spectrometer Science Investigation (PEPSSI) instrument on board the New Horizons spacecraft that flew by Pluto in 2015. Even though the measured ions have gyroradii larger than the size of Pluto and the cross section of its magnetosphere, we find that the boundary of the magnetosphere is depleting the energetic ion intensities by about an order of magnitude close to Pluto. The intensity is increasing exponentially with distance to Pluto and reaches nominal levels of the interplanetary medium at about 190R P distance. Inside the wake of Pluto, we observe oscillations of the ion intensities with a periodicity of about 0.2 hr. We show that these can be quantitatively explained by the electric field of an ultralow-frequency wave and discuss possible physical drivers for such a field. We find no evidence for the presence of plutogenic ions in the considered energy range.

An unusual cause of haemoptysis in childhood: A case report and literature review.

Haemoptysis is uncommon in children and the diagnosis is challenging. We describe a 14-year-old child who presented with haemoptysis secondary to a suspected congenital broncho-oesophageal fistula. This is a rare condition and the symptoms are insidious, occasionally beginning in childhood but may present only in adulthood. The case report describes the presentation, diagnosis and management of broncho-oesophageal fistulas, with a review of the current literature.

Acute high-altitude illness.

Letter by Basnyat on article by Hofmeyr et al. (Hofmeyr R, Tölken G, De Decker R. Acute high-altitude illness. S Afr J Med 2017;107(7):556-561. https://doi.org/10.7196/SAMJ.2017.v107i7.12612); and response by Hofmeyr et al.

Predicted v. real prevalence of the 22q11.2 deletion syndrome in children with congenital heart disease presenting to Red Cross War Memorial Children's Hospital, South Africa: A prospective study.

BACKGROUND: The 22q11.2 deletion syndrome (22qDS) has more than 180 associated phenotypic features, yet genotype-phenotype correlation remains obscure. Since many of the clinical characteristics are serious, yet treatable (including congenital heart disease), clinicians must maintain a high index of clinical suspicion to recognise a suite of co-occurring phenotypic features that suggest a diagnosis of 22qDS. Óskarsdottir's scoring schedule (the 'O score') is generally used to suggest the need for confirmatory fluorescent in situ hybridisation (FISH) testing, using the TUPLE 1 probe. An O score of two or more indicates the need for FISH testing. Objectives. A previous audit of FISH-positive results of patients with congenital heart disease at Red Cross War Memorial Children's Hospital (RCWMCH) revealed a clinical recognition rate of 1.7%. However, we were concerned that the syndrome may be under-recognised in our setting. Our aims were therefore to assess the predictive value of 'O scoring' and to accurately determine the prevalence of 22qDS in our patient population. Methods. A prospective trial of FISH testing every new patient with congenital heart disease presenting to RCWMCH was undertaken to accurately determine the prevalence of 22qDS. The results were then compared with the ability of the O score to indicate the need for FISH testing. RESULTS: Testing of 125 patients detected deletions in six (4.8%, 2.8 times the previously determined clinical detection rate), thereby vindicating our concern that 22qDS is under-diagnosed. Of these 125 patients, 37 had an O score of 2 or 3, yet only 6 were FISH-positive, giving the O score a positive predictive value of only 14%. Conclusion. Until a more robust alternative recognition tool is available, South African clinicians should use all clinical recognition criteria liberally to suggest the need for formal testing for 22qDS.

Excess BMP Signaling in Heterotopic Cartilage Forming in Prg4-null TMJ Discs.

Heterotopic cartilage develops in certain pathologic conditions, including those affecting the human temporomandibular joint (TMJ), but the underlying molecular mechanisms remain obscure. This is in part due to the fact that a reliable animal model of such TMJ diseases is not available. Here, we show that aberrant chondrocyte differentiation and ectopic cartilage formation occur spontaneously in proteoglycan 4 (Prg4) mutant TMJ discs without further invasive procedure. By 2 mo of age, mutant disc cells displayed chondrocyte transdifferentiation, accompanied by strong expression of cartilage master gene Sox9 and matrix genes aggrecan and type II collagen. By 6 mo, heterotopic cartilage had formed in the discs and expressed cartilage hypertrophic markers Runx2 and ColX. The ectopic tissue grew in size over time and exhibited regional mineralization by 12 mo. Bone morphogenetic protein (BMP) signaling was activated with the ectopic chondrogenic cells and chondrocytes, as indicated by phosphorylated Smad 1/5/8 nuclear staining and by elevated expression of Bmp2, Bmpr1b, Bmpr2, and BMP signaling target genes. Likewise, we found that upon treatment with recombinant human BMP 2 in high-density micromass culture, mutant disc cells differentiated into chondrocytes and synthesized cartilage matrix more robustly than control cells. Importantly, a specific kinase inhibitor of BMP receptors drastically attenuated chondrogenesis in recombinant human BMP 2-treated mutant disc cultures. Unexpectedly, we found that Prg4 was expressed at joint-associated sites, including disc/muscle insertion and muscle/bone interface, and all these structures were abnormal in Prg4 mutants. Our data indicate that Prg4 is needed for TMJ disc integrity and function and that its absence leads to ectopic chondrogenesis and cartilage formation in conjunction with abnormal BMP signaling. Our findings imply that the BMP signaling pathway could be a potential therapeutic target for prevention or inhibition of ectopic cartilage formation in TMJ disease.

Oro-facial pain and nutrition: a forgotten relationship?

Oro-facial pain (OFP) is known to exert profound impacts on quality of life including functionally and psychosocially mediated changes in dietary intake and thereby nutrition. This commentary explores the evidence base available on chronic oro-facial pain, diet and nutrition and discusses current dietary guidance for individuals with chronic OFP; potential impact of chronic OFP on eating and nutritional status; impact of nutritional status on pathophysiology of chronic OFP; and potential role of nutrition in the management of chronic OFP.

Local tunnel magnetoresistance of an iron intercalated graphene-based heterostructure.

The lateral variation of the tunnel magnetoresistance (TMR) of a graphene-based vertical heterostructure is studied by spin-polarized scanning tunneling microscopy (SP-STM) using an Fe-coated probe tip. The well-defined heterostructure is obtained by the intercalation of a magnetic Fe monolayer at the graphene/Ir(1 1 1) interface. Its structure is characterized by a moiré pattern with a high corrugation. In contrast to the Fe / Ir(1 1 1) surface, graphene/Fe / Ir(1 1 1) exhibits ferromagnetic order with an out-of-plane easy magnetization axis. At the nanometer scale, our experiments reveal that the moiré pattern induces a lateral variation of the TMR, which reaches 80%. The measured TMR at valleys of the moiré pattern is higher than at hills. We interpret this modulation in terms of a different hybridization between graphene and Fe at valleys and hills due to a different graphene-Fe distance at these sites, which leads to a different transmission of spin-polarized states.

Lubricin is Required for the Structural Integrity and Post-natal Maintenance of TMJ.

The Proteoglycan 4 (Prg4) product lubricin plays essential roles in boundary lubrication and movement in limb synovial joints, but its roles in temporomandibular joint (TMJ) are unclear. Thus, we characterized the TMJ phenotype in wild-type and Prg4(-/-) mouse littermates over age. As early as 2 weeks of age, mutant mice exhibited hyperplasia in the glenoid fossa articular cartilage, articular disc, and synovial membrane. By 1 month of age, there were fewer condylar superficial tenascin-C/Col1-positive cells and more numerous apoptotic condylar apical cells, while chondroprogenitors displayed higher mitotic activity, and Sox9-, Col2-, and ColX-expressing chondrocyte zones were significantly expanded. Mutant subchondral bone contained numerous Catepsin K-expressing osteoclasts at the chondro-osseous junction, increased invasive marrow cavities, and suboptimal subchondral bone. Mutant glenoid fossa, disc, synovial cells, and condyles displayed higher Hyaluronan synthase 2 expression. Mutant discs also lost their characteristic concave shape, exhibited ectopic chondrocyte differentiation, and occasionally adhered to condylar surfaces. A fibrinoid substance of unclear origin often covered the condylar surface. By 6 months of age, mutant condyles displayed osteoarthritic degradation with apical/mid-zone separation. In sum, lubricin exerts multiple essential direct and indirect roles to preserve TMJ structural and cellular integrity over post-natal life.

TMJ degeneration in SAMP8 mice is accompanied by deranged Ihh signaling.

The temporomandibular joint (TMJ) functions as a load-bearing diarthrodial joint during mastication, and its continuous use and stress can lead to degeneration over age. Using senescence-accelerated (SAMP8) mice that develop early osteoarthritis-like changes in synovial joints at high frequency, we analyzed possible molecular mechanisms of TMJ degeneration and tested whether and how malocclusion may accelerate it. Condylar articular cartilage in young SAMP8 mice displayed early-onset osteoarthritic changes that included reductions in superficial/chondroprogenitor cell number, proteoglycan/collagen content, and Indian hedgehog (Ihh)-expressing chondrocytes. Following malocclusion induced by tooth milling, the SAMP8 condyles became morphologically defective, displayed even lower proteoglycan levels, and underwent abnormal chondrocyte maturation compared with malocclusion-treated condyles in wild-type mice. Malocclusion also induced faster progression of pathologic changes with increasing age in SAMP8 condyles as indicated by decreased PCNA-positive proliferating chondroprogenitors and increased TUNEL-positive apoptotic cells. These changes were accompanied by steeper reductions in Ihh signaling and by expression of matrix metalloproteinase 13 at the chondro-osseous junction in SAMP8 articular cartilage. In sum, we show for the first time that precocious TMJ degeneration in SAMP8 mice is accompanied by--and possibly attributable to--altered Ihh signaling and that occlusal dysfunction accelerates progression toward degenerative TMJ disease in this model.

Search for the exit: Voyager 1 at heliosphere's border with the galaxy.

We report measurements of energetic (>40 kiloelectron volts) charged particles on Voyager 1 from the interface region between the heliosheath, dominated by heated solar plasma, and the local interstellar medium, which is expected to contain cold nonsolar plasma and the galactic magnetic field. Particles of solar origin at Voyager 1, located at 18.5 billion kilometers (123 astronomical units) from the Sun, decreased by a factor of >10(3) on 25 August 2012, while those of galactic origin (cosmic rays) increased by 9.3% at the same time. Intensity changes appeared first for particles moving in the azimuthal direction and were followed by those moving in the radial and antiradial directions with respect to the solar radius vector. This unexpected heliospheric "depletion region" may form part of the interface between solar plasma and the galaxy.

Structural and functional analysis of intra-articular interzone tissue in axolotl salamanders.

OBJECTIVE: Knowledge of mechanisms directing diarthrodial joint development may be useful in understanding joint pathologies and identifying new therapies. We have previously established that axolotl salamanders can fully repair large articular cartilage lesions, which may be due to the presence of an interzone-like tissue in the intra-articular space. Study objectives were to further characterize axolotl diarthrodial joint structure and determine the differentiation potential of interzone-like tissue in a skeletal microenvironment. DESIGN: Diarthrodial joint morphology and expression of aggrecan, brother of CDO (BOC), type I collagen, type II collagen, and growth/differentiation factor 5 (GDF5) were examined in femorotibial joints of sexually mature (>12 months) axolotls. Joint tissue cellularity was evaluated in individuals from 2 to 24 months of age. Chondrogenic potential of the interzone was evaluated by placing interzone-like tissue into 4 mm tibial defects. RESULTS: Cavitation reached completion in the femoroacetabular and humeroradial joints, but an interzone-like tissue was retained in the intra-articular space of distal limb joints. Joint tissue cellularity decreased to 7 months of age and then remained stable. Gene expression patterns of joint markers are broadly similar in developing mammals and mature axolotls. When interzone-like tissue was transplanted into critical size skeletal defects, an accessory joint developed within the defect site. CONCLUSIONS: These experiments indicate that mature axolotl diarthrodial joints are phenotypically similar to developing synovial joints in mammals. Generation of an accessory joint by interzone-like tissue suggests multipotent cellular differentiation potential similar to that of interzone cells in the mammalian fetus. The data support the axolotl as a novel vertebrate model for joint development and repair.

Bacillus anthracis comparative genome analysis in support of the Amerithrax investigation.

Before the anthrax letter attacks of 2001, the developing field of microbial forensics relied on microbial genotyping schemes based on a small portion of a genome sequence. Amerithrax, the investigation into the anthrax letter attacks, applied high-resolution whole-genome sequencing and comparative genomics to identify key genetic features of the letters' Bacillus anthracis Ames strain. During systematic microbiological analysis of the spore material from the letters, we identified a number of morphological variants based on phenotypic characteristics and the ability to sporulate. The genomes of these morphological variants were sequenced and compared with that of the B. anthracis Ames ancestor, the progenitor of all B. anthracis Ames strains. Through comparative genomics, we identified four distinct loci with verifiable genetic mutations. Three of the four mutations could be directly linked to sporulation pathways in B. anthracis and more specifically to the regulation of the phosphorylation state of Spo0F, a key regulatory protein in the initiation of the sporulation cascade, thus linking phenotype to genotype. None of these variant genotypes were identified in single-colony environmental B. anthracis Ames isolates associated with the investigation. These genotypes were identified only in B. anthracis morphotypes isolated from the letters, indicating that the variants were not prevalent in the environment, not even the environments associated with the investigation. This study demonstrates the forensic value of systematic microbiological analysis combined with whole-genome sequencing and comparative genomics.

The complete genome sequence of Bacillus anthracis Ames "Ancestor".

The pathogenic bacterium Bacillus anthracis has become the subject of intense study as a result of its use in a bioterrorism attack in the United States in September and October 2001. Previous studies suggested that B. anthracis Ames Ancestor, the original Ames fully virulent plasmid-containing isolate, was the ideal reference. This study describes the complete genome sequence of that original isolate, derived from a sample kept in cold storage since 1981.