Post-cataract macular edema (PCME) is a condition that can occur in patients following cataract surgery without risk factors and complications. Although 80% of patients experience spontaneous resolution after 3 to 12 months, in persistent cases, it can lead to permanent vision loss if left untreated. There are currently no standardized treatment guidelines for PCME, and there have been limited studies showing the impact of PCME on annual Medicare spending and ophthalmology-related outpatient visits per case compared to those without the complication. This study aims to evaluate real-world treatment patterns and the economic burden of patients with PCME.® MarketScan® Commercial and Medicare Supplemental databases. Patients with (n = 2430) and without (n = 7290) PCME 1 year post cataract surgery were propensity score matched 1:3 based on age, geographic region, diabetes presence, cataract surgery type, and Charlson Comorbidity Index. Treatment pattern analysis for each PCME patient summarized the distribution of medications across lines of therapy. Economic burden analysis compared the mean number and costs of eye-related outpatient visits, optical coherence tomography imaging scans, and ophthalmic medications between the 2 groups using linear regression models.This retrospective claims analysis identified patients from the IBMTreatment pattern analysis found 27 different treatment combinations across 6 treatment lines. The most common first-line treatments were topical steroid drops (372 [30%]), topical nonsteroidal anti-inflammatory drug drops (321 [27%]), and intraocular or periocular injectable steroids (189 [15%]). Compared to match controls, PCME patients averaged 6 additional eye-related outpatient office visits (95% CI: 5.7–6.2) resulting in an additional $3,897  in total costs. Patients filled 3 more ophthalmology-related outpatient prescription medications (95% CI: 2.8–3.2), adding $371 in total cost (95% CI: $332 – $410).PCME treatment patterns showed wide clinical variability in treatments and time, specifically regarding injectable treatments and combination therapy. Additionally, significantly higher healthcare resource use and economic burden were found for both patients and payers when comparing PCME patients to non-PMCE controls. These results highlight the need for treatment standardization and demonstrate that interventions targeted at preventing PCME may be valuable.The online version contains supplementary material available at 10.1186/s12886-023-03113-x. Cataract surgery is one of the most common ocular surgeries, with an estimated 3.6 million surgeries performed each year in the United States . The preThere are currently no uniform diagnosis criteria for PCME . Common PCME is self-limiting with 80% of patients experiencing spontaneous resolution after 3–12 months , 6. HoweDue to the large volume of cataract surgeries performed each year, PCME management can add up to sizable costs. One study reported that PCME increased Medicare ophthalmic payments by 85% and doubled ophthalmic charges from $5,950 to $10,410 (in 2016 US dollars) . AnotherThere have been no studies to date assessing treatment patterns and few studies characterizing the economic consequences of PCME; therefore, this retrospective, real-world analysis of claims data aimed to evaluate the distributions of medications used for each line of therapy to depict treatment patterns for PCME and quantify the economic burden.® MarketScan® Commercial and Medicare Supplemental databases. This dataset represents ~ 40 million commercially insured US adults or US adults qualifying for Medicare that have commercial supplemental insurance. The dataset is de-identified and compliant with the Health Insurance Portability and Accountability Act of 1996. The analysis was considered non-human subjects research by the Institutional Review Board (IRB) at the University of Washington and IRB approval was not required.Administrative claims data was used from the IBMA retrospective claims analysis was performed to identify patients aged 18 years or older who had cataract surgery between 2014 and 2017. The first cataract surgery date served as each patient’s index date and was identified using Current Procedural Terminology (CPT) codes. Sample enrollment was limited to patients who had ≥ 1 year of continuous enrollment prior to their index and ≥ 3 years of continuous enrollment post index. International Classification of Diseases 9th and 10th Revision (ICD-9 and ICD-10) codes for PCME were used to separate patients into PCME and non-PCME groups. Patients in the PCME group were matched 1:3 to each non-PCME case using logistic regression to estimate propensity scores. Detailed methods for propensity score matching and a list of all procedure and diagnosis codes can be found in the Supplemental Materials . In the PCME group, the PCME diagnosis date served as the event date which had to occur within one year from the index date. A 1-year period was selected to ensure time for the development of PCME from cataract surgery, while accounting for flexibility in timing due to the real-world nature of the study. Time > 1 year was excluded since it is unlikely that macular edema that develops at this time point is due to cataract surgery. The number of post-operative days between the index and event date was used to determine an equivalent event date for each matched control. The full dataset contained dates between January 1, 2013, and December 31, 2019. Baseline clinical and demographic data were assessed during the year prior to or on each patient’s index date. Treatment patterns and economic burden were assessed throughout the 2-year follow-up period after each patient’s event date  score . OutpatiRelevant PCME-related medications following the diagnosis event date through the 2-year follow-up period were reviewed. PCME-related treatment was defined as treatments given up to 3 months from the last outpatient service claim containing a PCME diagnosis. Included treatments consisted of medications used for PCME based on current literature  , 10. NSAMean counts and costs over the entire group were determined and used to provide per patient estimates. Healthcare resource utilization (HCRU) included the number of unique eye-related outpatient office visits (any visit with an eye-related provider or visits containing a PCME diagnosis code), OCT scans, and ophthalmology-related medications (number of relevant prescription medication fills and injectable treatments administered) accumulated by PCME and non-PCME patients. The number, mean, and percentage of patients who had ≥ 1 claim was recorded. Patient-related costs were those incurred by the patient and consisted of the sum of the patient co-pay, co-insurance, and deductibles. Payer-related costs were total payments made by the health plan to healthcare providers and total costs were the sum of patient and payer costs. All costs were adjusted to 2022 US dollars using the medical care component of the Consumer Price Index for all urban consumers . A subgrt-tests for continuous variables, and Chi-squared or Fischer’s exact tests for categorical variables. Differences in the mean number and costs of eye-related outpatient visits, OCT scans, ophthalmic prescription medications, and injectable medication claims were assessed using multivariable linear regression models that adjusted for age, geographic region, diabetes presence, complex cataract surgery, number of cataract surgeries, and CCI score . A sensitivity analysis using the cataract surgery date as the index date was performed for mean counts and costs . SAS version 9.4  was used for constructing the analytical dataset and all analyses were conducted in RStudio version 1.4.1106 . A 2-sided alpha with a significance level of 5% was used for all statistical comparisons.Baseline characteristics were summarized for all variables using mean and standard deviation (SD) for continuous variables, and frequencies and proportions for categorical variables. Differences were assessed using Student’s P = .04). Patients in the PCME group had a significantly higher prevalence of PCME risk factors (P < .001). There were no other statistically significant differences. The mean time from cataract surgery to PCME diagnosis was 3.6 ± 2.9 months, and 295 (12.1%) patients were found to have persistent PCME . In total, 1,222 patients received ≥ 1 line of therapy. Of these individuals, 411 34%) advanced to a second-line treatment and 47 (3.9%) received treatment beyond the fourth line. Regimens containing an EA NSAID were slightly more common than regular NSAIDs (456 vs. 373). NSAIDs were given more frequently as combination therapy with a topical steroid whereas EA NSAIDs were given more frequently as monotherapy. Otherwise, no major differences between the 2 types were observed and both were analyzed together as a class during analysis. Most patients received monotherapy  or dual therapy (199 [16%]) first line and this trend continued across all treatment lines. The most common first-line treatments were monotherapies of topical steroids (372 [30%]), NSAIDs as a class (321 [27%]), and injectable steroids (189 [15%]). Ocular injectable containing regimens comprised 378 (31%) of first-line treatments. The most common second-line treatments included monotherapy of NSAIDs as a class (96 [24%]), dual therapy of any NSAID and topical steroid (77 [19%]), and topical steroid monotherapy (72 [18%]). Treatment sequencing across all 6 lines of therapy for any treatment line with > 1 count is shown in Fig. 4% advancP < .0001) (Table The PCME group accumulated more HCRU (in terms of counts and costs) and had a higher proportion of patients with ≥ 1 claim across all categories compared to match controls. More patients in the PCME group had ≥ 1 eye-related outpatient provider visit compared to the control group , leading to an excess of 6 visits (95% CI: 5.7–6.2). Claims for ≥ 1 OCT scan occurred in 95% of the PCME group compared to 17% of the control group, leading to an excess of 4.5 scans (95% CI: 4.3–4.7). The PCME group received an excess of 3 prescription medications (95% CI: 2.8–3.2) and 0.9 injectable medications (95% CI: 0.7–1.0) compared to the control group. All differences were statistically significant (P < .0001)  costs (95% CI: $341 - $419) and $3,517 in payer costs , equaling to a mean adjusted incremental total difference of $3,897 . The difference in adjusted total costs for OCT was $295 (95% CI: $268 - $322) higher for the PCME group. Patients and payers also spent more on prescription and injectable medications which resulted in higher mean adjusted costs for both categories . All differences were statistically significant , topical NSAIDs as a class (108 [20%]), and dual therapy with both (91 [17%]). Additionally, most patients who received triple therapy or advanced to a sixth treatment line did not receive prophylactic therapy. There were no statistically significant differences in counts or costs of resources used between PCME patients who received prophylactic therapy and those who did not .This retrospective claims analysis evaluated the real-world treatment patterns and economic burden of cataract surgery patients who developed PCME compared to those who did not. PCME treatment patterns showed wide variability in the approaches to treatment and a significant economic burden for both patients and payers in the PCME group was identified.The literature describes PCME treatment as a stepwise approach starting with topical monotherapy, then topical dual therapy, and eventually periocular or intraocular injectables as last-line options in prolonged or non-responsive cases , 10. HowTwo previous studies have explored the relationship between PCME and costs; both reported a similar directionality of outcomes to this study but were limited in sample size and follow-up time , 13. ThiA significantly higher proportion of patients in the control group compared to the PCME group had received prophylactic therapy and the subgroup analysis of PCME patients who used prophylactic therapy found that these patients were less likely to receive injectable treatments or advance to a sixth-line treatment. Surprisingly, comparisons of the economic burden between PCME patients who did and did not receive prophylactic therapy failed to result in any significant findings. This suggests that although prophylactic therapy may be effective at reducing the risk of PCME, once PCME develops, it may have minimal impact on reducing the long-term severity or intensity of the condition measured by incremental HCRU. Despite the latter, emphasis on prevention in clinical practice is imperative to avoid the consequences of PCME. NSAIDs have been shown to be effective in preventing PCME through their anti-inflammatory effects and ability to improve surgical efficiency by preventing pupillary miosis , 20. Pro® database which represents individuals who have Medicare supplemental or commercial insurance. This could impact the generalizability of the results for those who are aged 65 years or older, which is the group that is more commonly affected by cataracts and most at risk for developing PCME. Additionally, because this is a claims dataset, it was not possible to confirm which eye was being treated, which may have resulted in the misclassification of treatments being grouped as combination therapy and the introduction of bias to the results. Another potential limitation was the 3 years of post-index enrollment required for inclusion and the long duration of follow-up used for the analysis. This limited the sample size and could have led to the incorporation of treatments and resources that were no longer related to PCME. The long follow-up period was chosen to account for patients who had persistent PCME and to capture any potential long-term consequences of the disease since it has been reported that patients may still experience visual consequences from PCME after resolution [A potential limitation to this study is the Marketscansolution . HoweverCataract surgery is one of the most frequently performed surgical procedures worldwide, making PCME a condition that can result in significant clinical and economic burden. This study found wide variability in treatment regimens used, especially with combination therapy and injectable treatments, highlighting the need for treatment standardization. These results also show that those who develop PCME following cataract surgery incur higher costs and HCRU compared to those who did not, demonstrating that interventions targeted at preventing PCME may be valuable. The need for high-quality evidence from randomized controlled trials is essential as well as the creation of best practices for the treatment of PCME to reduce variability and waste. Future studies should explore the treatment patterns and economic burden of PCME and incorporate data from sources that can provide specific details , such as electronic health records.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2Supplementary Material 3Supplementary Material 4Supplementary Material 5


Mycobacterium tuberculosis. Tuberculosis continues to be a major public health problem globally. Mortality rates have not decreased significantly; rather, they are increasing. This review has thus been aimed at deepening knowledge regarding tuberculosis by examining published material related to an immune response against Mycobacterium tuberculosis, mycobacterial evasion mechanisms regarding such response and the relationship between pulmonary and extrapulmonary clinical manifestations induced by this bacterium which are related to inflammation associated with tuberculosis dissemination through different routes.Tuberculosis is a far-reaching, high-impact disease. It is among the top ten causes of death worldwide caused by a single infectious agent; 1.6 million tuberculosis-related deaths were reported in 2021 and it has been estimated that a third of the world’s population are carriers of the tuberculosis bacillus but do not develop active disease. Several authors have attributed this to hosts’ differential immune response in which cellular and humoral components are involved, along with cytokines and chemokines. Ascertaining the relationship between TB development’s clinical manifestations and an immune response should increase understanding of tuberculosis pathophysiological and immunological mechanisms and correlating such material with protection against  Mycobacterium tuberculosis (Mtb) bacillus which is found in the MTB complex; it is principally transmitted through aerosols that are expelled from a person having active TB. Efforts at slowing the disease’s progression have involved studies in fields related to epidemiology, risk factors, immune response, TB pathophysiology, new diagnostic and therapeutic tools for all forms of infection and the disease itself  for centuries; it is an infectious bacterial disease and has become positioned as one of the main causes of mortality worldwide. This infection is mainly caused by the Mtb can spread from an initial source of infection via different routes and can affect almost all the body’s organs. A 2021 estimate showed that around 10.6 million people had become ill with TB, representing an increase from 10.1 million in 2020; incidence rate increased by 3.6% between 2020 and 2021 and 1.4 million cases occurred. Regarding TB-related deaths, 57% of cases were men, 33% women and 11% children  and phagocytic cells (neutrophils (N), monocytes (M) and dendritic cells (DC). Infection does not occur if such first line of defence succeeds in rapidly eliminating Mtb, otherwise phagocytes become infected and Mtb reproduces within these cells, initially causing few if any clinical manifestations  during primary infection or during TB reactivation later in life. EPTB can involve any host body organ, the lymph nodes being the most common site; however, pleural, neurological, osteoarticular and/or genitourinary involvement have also been described, each having a particular clinical presentation associated with an acute or chronic inflammatory response triggered by the immune system to destroy tb Fogel .Mtb, its evasion mechanisms used in the attempt to survive host immune system defence and the clinical manifestations of TB related to pulmonary and extrapulmonary involvement, summarised in Fig. This review discusses the innate and adaptive immune response triggered by Mtb enters a host when active TB patients’ microdroplets are inhaled (i.e. in which the bacilli are suspended), reaching the bronchial trees where bacteria come into contact with the respiratory mucosa; this is coated with airway surface liquid (ASL) in which there is mucus, antimycobacterial peptides, immunoglobulins, cytokines and chemokines which try to prevent the microorganism’s passage  containing Mφ and mucosal-associated invariant T-cells (MAIT) which produce IFN-γ, TNF-α and granzyme used for counteracting b Lugton .Mtb cell wall. The Natural Killer cells (NK) become extravasated; these mediate cell cytotoxicity through degranulation induced by IL-2 and cytokine signalling like IFN-γ (induced by IL-12). It has been shown that IL-12-activated NK cells can inhibit Mtb and Mycobacterium avium growth and can also develop memory by exposure to antigens, thereby establishing a bridge between innate and adaptive responses , produce IL-22 that inhibits mycobacterial intracellular growth and express receptors for soluble factors such as cytokines  which produce surfactant antimicrobial substances, such as hydrolases, that affect es Denis . It has Mtb via phagocytosis, leading to exacerbation of tissue inflammation and damage , promoting TL-, BL- and NK cell-mediated immunity and Mφ and DC recruitment via chemokines. DCs migrate to lymph nodes for mycobacterial antigen presentation to more lymphocytes; however, polymorphonuclear cells may be unable to clear age Fig. .Mtb and thus purified protein derivative (PPD) or IFN-γ release tests will prove negative. However, it has been well documented that mycobacteria can proliferate due to their immune response evasion mechanisms (as widely reported regarding Mφ)  recognition of the bacillus orchestrates monocyte migration in peripheral blood to the infection site and their differentiation into Mφ; Mφ phagocytosed bacilli can die in the phagolysosome.Mtb, given that memory and effector cells are produced which can induce the lysis of infected Mφ, along with contributing to their activation by means of IFN-γ for eliminating mycobacteria , increased HLA-II expression, the promotion of apoptosis and autophagy , having strong affinity for various antigens. Follicular helper T-cells (Tfh) play an important role in GC formation. These cells express the CXCR5 receptor enabling BL migration to the CG; their formation requires an inducible T-cell COStimulator (ICOS). Tfh also produce IL-21 which (together with CD40L) promotes BL differentiation and isotype switching for producing long-lasting Ab-producing memory and plasma cells  and inhibition of autophagy, associated with mycobacterial intracellular degradation  and ESAT-6, which could contribute to the pathogenesis of the microorganism causing the infection  are involved in inhibiting Tregs are induced by mycobacterial antigens in lymph nodes early on during infection, thus delaying effector TL activation by releasing IL-10 and transforming growth factor β (TGF β) ) has been used for studying the functional effect of MAIT cells expressing programmed death receptor 1 (PD-1) in TB patients. It was shown that MAIT cells expressing PD-1 reduced IFN-γ levels; however, CXCL13 and IL-21 levels increased, even though such cells’ presence was more related to an extension of the infection  TCR. Mycobacterial antigens also come into contact with these lymphocytes (1–5% of circulating T-lymphocytes) triggering cell-mediated immune responses , in addition to HLA class I and II antigen presentation. Such mechanism involves mycobacterial glycolipids which are abundant in the cell wall, such as phosphatidylinositol mannosides, lipoarabinomannan, mycolic acids and sulfoglycolipids, such presentation usually induces IFN-γ production.It has been found that CD1a is expressed on cell surface and in early endosomes whilst CD1b is expressed in late endosomes or lysosomes. CD1c and CD1d are located on cell surfaces and are recycled in late endosomes. These molecules are abundant in DCs; however, they are downregulated in mycobacteria-infected cells. CD1d is expressed in haematopoietic and non-haematopoietic cells, including epithelial cells Kaufmann .Mtb, along with cytotoxic granule expression, have been observed following pathogen challenge when Mtb-specific CD1b T-lymphocytes have been transferred  and TB development is facilitated in hosts having a deficiency regarding Ab or B-lymphocyte production , lipoglycoproteins and polysaccharides located on cell envelope; they also recognise proteins such as heparin-binding haemagglutinin (HBHA) and the Mtb 16kDA protein (HspX)  had the opposite effect, promoting infection , it was established via intracellular growth inhibition assays in Mφ cell lines that the IgG anti-AM (arabinomannan) produced naturally during asymptomatic individuals’ (TBL) infection was protective, but not in sick donors (TBA)  intravenously immunised with BCG had IgM Ab titres which correlated significantly with a decrease in Mtb load in the lungs. Mtb-specific IgM monoclonal Ab (mAb) were obtained that reduced pathogen in vitro survival  interact with mycobacterial ligands. Such inflammatory elements are responsible for recruiting new cells at the primary infection site and also trigger granuloma formation by the innate immune system. The adaptive immune response usually occurs 4 to 6 weeks after DC antigen presentation in regional lymph nodes  can be created through the classical pathway (involving Ab action) through which mycobacterial lysis is induced  represent another mechanism capable of inducting trained immunity; like BCG it induces different gene expression patterns resulting in the elimination of invading pathogens  are haphazardly recruited to an infection site, thereby exacerbating inflammation and thus increasing disease severity  containing one to five Mycobacteria induce Mφ necrosis and the recruitment of more Mφ and myeloid cells, thereby enabling further replication to occur . Symptoms are constitutional and pulmonary, their frequency depending on whether the patient has primary TB or reactivated TB. Subjects having primary TB are much more likely to be asymptomatic or minimally symptomatic , reaching more than 50% of cases. Active TB presentation can be highly variable, ranging from a state in which there are no symptoms to severe associated sequelae, such as Pott’s disease, or even become life-threatening, as in the case of tuberculous meningitis  and pulmonary hilum lymph nodes so involved to which the affected area drains. Bacilli entering the bloodstream or lymphatic system spread, resulting in extrapulmonary TB during primary infection or later on during reactivation of the disease. The lymph nodes are the most commonly affected site by extrapulmonary TB; however, pleural, neurological, synovial, musculoskeletal, pericardial, peritoneal and genitourinary involvement have also been described  was able to isolate the e Duarte . This clSuch clinical picture constitutes about 35% of extrapulmonary TB cases; the most commonly affected body area is the cervical region, accounting for 60–90% of reported cases. Cervical lymph node involvement is due to lymphatic dissemination of bacilli from the Ghon complex or from the tonsils, adenoids or from involved ethmoid bone. Once mycobacterial colonisation occurs, the bacilli proliferate inside the lymph nodes causing local inflammatory changes, such as marked hyperaemia, oedema, necrosis and caseation of compromised lymph nodes.Inflammatory changes spread regionally in the area of the involved ganglion chain following the spread of bacilli to adjacent nodes causing adjacent skin to adhere or nodes to rupture into surrounding tissues or through the skin, forming sinuses or fistulous tracts. Compromise of the mediastinal ganglion chains can cause compression of structures, such as major blood vessels and the recurrent phrenic or laryngeal nerves, or even cause bronchial. The detailed mechanisms of bacterial spread remain unclear. Bacilli must traverse the alveolar epithelium to reach draining lymph nodes and the bloodstream. It has been shown that mycobacteria within alveolar Mφ or DCs can be relocated to lymph nodes and blood and that mycobacteria can also invade and lyse epithelial cells after infecting them  and endothelial cell adhesion molecule expression (CD54), leading to cell accumulation in inflammatory foci. Type II interferons (IFN-γ) have been the most studied cytokines regarding anti-tuberculous immunity due to their great importance for phagocytosis and subsequent mycobacterial death. IFN-γ is mainly produced by activated CD4+ and CD8+ T-cells and, to a lesser extent, by γδ T-cells, NK T-cells and innate immunity-related NK cells.The strongest IFN-γ gene expression is detected in activated Th1-cells stimulating Mφ to eliminate mycobacteria, enhance other cells’ cytotoxic activity, induce apoptosis in skin and mucosa epithelial cells, regulate MHC class I and II protein expression and antigen presentation. IL-10 is an inflammatory response multifunctional regulatory cytokine and has been shown to act as a general inhibitor of Th1 and Th2 cell proliferative responses. IL-10 regulates inflammation by suppressing the production of cytokines such as IL-1α, IL-1β, IL-6, IL-8, IL-12 and TNF-α in activated Mφ and IFN-γ in T-cells. TGF-β is the main representative of the family which currently consists of 35 factors, including 5 TGF-β isoforms, bone morphogenic protein (BMP), growth differentiation factors (GDF) and activin and inhibin factors. TGF-β is secreted in an inactive form (L-TGF-β); it is activated after the action of plasmin, thrombospondin-1, reactive oxygen radicals and αVβ6 integrin.Concerning TB, antigen-regulatory antigen-specific Th1 is the main IL-10 producer and Th3 the main TGF-β one; they are activated upon cooperation with DCs and produce optimal interaction balance with specific effector T-cells and control tuberculosis infection regarding an excessive immunopathological response  release  in pleural fluid , identifying a negative association between the percentage of lymphocytes in pleural fluid and the probability of a positive effusion culture result (suggesting infection and clearance) and higher mycobacterial load in loculated effusions. The initial response to mycobacteria in the pleura has been shown to consist of a rapid influx of polymorphonuclear leukocytes, particularly neutrophils which remain in predominant cells for the first 24 hours, followed by Mφ, which peak after 96 hours, and lymphocytes , determining granulomas in biopsy of the pleura or ADA levels being determined in a suitable context . Such foci can break into the subarachnoid space, triggering a T-cell response having high IFN-γ and TNF-α levels in cerebrospinal fluid  disease caused by mycobacteria is among the least common, but most lethal forms of  Leonard .CNS infection comprises a spectrum covering subacute or chronic meningitis, intracranial tuberculoma and tuberculous spinal arachnoiditis. All three forms can be observed in regions of high TB prevalence where there is clinical post-primary extrapulmonary infection among children and young adults. Tuberculous meningitis initially involves haematogenous mycobacterial dissemination from a primary pulmonary focus or late reactivation in another body region. The blood-brain barrier is not breached in such mycobacteremia to produce immediate direct invasion of the meninges and subarachnoid space, but rather a scant number of bacilli spread throughout the brain substance, meninges and adjacent tissues, causing the formation of multiple, small, granulomatous foci having different sizes and degrees of encapsulation which may coalesce to form larger caseous foci. If such foci are adjacent to the ependyma or pia mater they may eventually rupture into the subarachnoid space, causing meningitis Leonard .CNS TB thus occurs in sustained post-primary bacillaemia conditions  and those involving a lack of sustained immunological control of latent foci in the brain or other body regions (as in the elderly and other immunosuppressed or HIV-infected adults). Granulomatous focus rupture into the subarachnoid space triggers an intense cytokine-mediated inflammatory reaction that becomes more marked at the base of the brain, causing basal proliferative arachnoiditis, vasculitis or hydrocephalus. Direct mycobacterial invasion of the blood vessel wall, or secondary extension of adjacent inflammation, leads to an intense polymorphonuclear reaction within the tunica adventitia vascularis, followed by lymphocyte, plasma cell and Mφ infiltration. Progressive destruction of the adventitia enables the inflammatory process to reach the tunica intima vascular, causing aneurysms, thrombosis and haemorrhage , weight loss, diaphoresis, headache, seizures, progressive hemiplegia and/or signs of increased intracranial pressure Garg .The rupture of a focus within the spinal cord or meninges, or extension of an adjacent area of spondylitis, may be associated with the development of arachnoiditis or tuberculoma at any level of the spinal cord. The triggered inflammatory reaction is usually limited to the involved region and can gradually progress over weeks to months, leading to the appearance of a gelatinous or fibrous mass partially or totally covering the spinal cord. Clinical manifestations are associated with compression of the nerve root and spinal cord, secondary to impingement by advancing arachnoiditis. Clinical manifestations are predominantly neurological and include pain, hyperesthesia or paraesthesia in nerve root distribution, lower motor neuron palsy and loss of sphincter control Leonard .All urinary and reproductive tract tissues become infected during mycobacterial extrapulmonary seeding, occurring in 2–20% of cases. Any part of the tract may become involved; however, the most frequent involvement involves the kidneys, ureters, bladder, prostate and ovaries. The Mycobacterium’s slow replication rate, intracellular location in Mφ and acquired immune response mean that the symptoms and signs of infection take between 12 months and 2 years after primary infection for urogenital disease to manifest itself. Constant chronic interaction between the mycobacteria and host immune response can lead to eradication of the microorganism, or disease progression manifesting as caseous necrosis, miliary disease and the formation of abscesses, cysts, ulcers and fistulas, fibrosis or calcifications. Cell-mediated immune responses become reduced when immunosuppression occurs (HIV infection), enabling tubercle bacilli proliferation, causing a more severe clinical picture that progresses rapidly. Mycobacterial seeding occurs in various parts of the urogenital tract through haematogenous or lymphatic spread from a primary pulmonary or intestinal tuberculous focus  described paraplegia due to the destruction of the anterior vertebral column and progressive kyphosis. Pott’s disease is then defined as the presence of tuberculous spondylodiscitis that may or may not be accompanied by psoas abscess  , the current TB vaccine pipeline includes 29 candidate vaccines in different stages of clinical development  is a research and innovation partnership that facilitates TB vaccine discovery and development and works through the Global TB Vaccine Partnership (GTBVP). According to TBVI data , which has had good results in the preclinical phase  or as a novel replicating-RNA (repRNA) formulated in a nanostructured lipid carrier. Protein subunit- and RNA-based vaccines preferentially elicit cellular immune responses targeting different ID91 epitopes; both platforms reduced pulmonary bacterial burden in a single prophylactic immunisation screen, compared to controls. Excitingly, prime-boost strategy groups that received heterologous RNA-prime, protein-boost or combination immunisations had the greatest reduction in bacterial burden and a unique humoral and cellular immune response profile. Such data represents the first report that repRNA platforms are a viable system for TB vaccines and should be pursued using high-priority An mRNA-based vaccine can thus be developed for preventing TB; however, in practice this will depend on identifying the most appropriate antigen targets (which has been difficult to achieve to date). The WHO’s new mRNA technology transfer hubs have raised researchers’ optimism that the time has come to find a new anti-TB vaccine. The WHO experts consider that mRNA technology transfer hubs would empower low- and middle-income countries to produce their own vaccines, medicines and diagnostics, and the technology could also be used for developing TB vaccines. The mRNA technology has progressed over the years and actually leap-frogged during the COVID-19 pandemic; it is thus the right time to use mRNA technology for developing a new and effective TB vaccine.TB is the oldest documented infectious disease in humans, infecting almost a third of the world’s population. The host-microbe interaction is responsible for a broad clinical and pathological spectrum. Understanding TB immunology is essential for developing new diagnostic and therapeutic tools and forms the basis for developing an effective anti-TB vaccine.Research into this disease facilitates further understanding of the mycobacteria’s complex nature as it affects the usual host cell functions. It can directly inhibit the main immune system, as seen above; host defence system has a double-branched arsenal against mycobacteria, i.e. innate and adaptive immunity. Taken together, this leads to pathogen elimination and preventing disease development. Trained immunity should not be ignored as it also seems to be responsible for humans’ partial protection against mycobacteria.Mtb growth, although it is now thought that mycobacteria may hijack granulomas for their own advantage, thereby altering the immune response and enabling their dissemination via haematogenous, bronchogenic and lymphatic pathways from an initial pulmonary focus.The host immune system employs a broad spectrum of mechanisms to stop tubercle bacillus advance and pathogenesis. However, the pathogen has developed its own defence system, directly focusing on evading our immune response and thereby leading to the development of the clinical manifestations described in this review. Granuloma formation may control Mtb induces a new acute local immune response upon reaching the different organs and tissues after its dissemination; this could become chronic and its cellular inflammatory changes and cytokine and inflammation mediator production will cause the histopathological changes and systemic effects which are often not organ-specific, making TB the “great clinical simulator”. Clinical suspicion is thus highly relevant when arriving at diagnostic suspicion regarding the certainty of TB in its many forms, followed by timely establishment of suitable treatment, as necessary, according to host organs compromised by the tuberculosis bacillus.TB continues to be one of the twenty-first century’s most important public health problems; clinical and scientific advances have increasingly led to greater preventive, pathophysiological, immunological, diagnostic and therapeutic knowledge regarding all forms and clinical presentations of this disease. Collaborative multinational efforts must be continued to have an ever deeper and clearer conceptual understanding of TB that will enable it to be eradicated. Such collaborative efforts should include researchers, public health workers, health care staff and the general population with the sole purpose of expanding current knowledge and mitigating TB’s global burden.


When O2 levels in the alveoli and/or blood are low, compensatory adaptive reflexes are produced that increase the uptake of O2 and its distribution to tissues within a few seconds. This paper analyzes the most important acute vasomotor responses to lack of O2 (hypoxia): hypoxic pulmonary vasoconstriction (HPV) and hypoxic vasodilation (HVD). HPV affects distal pulmonary (resistance) arteries, with its homeostatic role being to divert blood to well ventilated alveoli to thereby optimize the ventilation/perfusion ratio. HVD is produced in most systemic arteries, in particular in the skeletal muscle, coronary, and cerebral circulations, to increase blood supply to poorly oxygenated tissues. Although vasomotor responses to hypoxia are modulated by endothelial factors and autonomic innervation, it is well established that arterial smooth muscle cells contain an acute O2 sensing system capable of detecting changes in O2 tension and to signal membrane ion channels, which in turn regulate cytosolic Ca2+ levels and myocyte contraction. Here, we summarize current knowledge on the nature of O2 sensing and signaling systems underlying acute vasomotor responses to hypoxia. We also discuss similarities and differences existing in O2 sensors and effectors in the various arterial territories.An adequate supply of oxygen (O Resistance pulmonary arteries constrict in response to a decrease in alveolar PO2 , thereby diverting blood flow from poorly ventilated lung regions towards areas with a higher PO2 to optimize ventilation/perfusion ratio. In conditions of persistent hypoxemia (low blood PO2), hypoxic pulmonary vasoconstriction (HPV) contributes to the pathogenesis of pulmonary hypertension (2-deprived tissues (Blood oxygen tension (POculation . Arteriertension . Unlike  tissues .2 sensor in vascular smooth muscle (VSM)  can modulate vessel diameter, numerous studies have nevertheless demonstrated the existence of endothelium-independent hypoxic vasomotor responses and suggested the existence of an acute Ole (VSM) . Indeed,lar tone . In thisvia the measurement of the isometric force exerted by isolated pulmonary arteries  also constrict in response to hypoxia. In contrast, hypoxia induces vasodilation in the proximal conducting vessels (main trunk and first branches of the pulmonary artery), as it does in the systemic vasculature. HPV is rapidly and reversibly triggered within a few seconds in response to hypoxia . The vasarteries  , resulting in a subsequent increase in vascular tone. In what follows, we briefly summarize the sensing/signaling systems reported so far which, although subject to debated, seem to have garnered experimental support.The HPV reflex implies the presence of a functional acute O2+ from intracellular stores via cyclic ADP-ribose  through its interaction with ryanodine receptors (RyR) in the sarcoplasmic reticulum. Although it is not yet established whether AMP kinase directly elicits cADPR-dependent Ca2+ release, it has been proposed that this could result from phosphorylation by AMP kinase of ADP-ribosyl cyclase, which catalyzes the cyclization of NAD+ to produce cADPR, and a subsequent increase in cADPR accumulation. AMP kinase could also increase the sensitivity of the Ca2+ release process to cADPR due to the phosphorylation of RyRs or an intermediate cADPR-binding protein  is capable of producing reactive oxygen species (ROS) in proportion to PO2 sensor . However2 sensor . Proteintivation . In thistraction . Howevertraction . In all 2+ sensitization, capable of inducing PASMC contraction by inhibiting myosin light-chain phosphatase in a Ca2+-independent manner  triggered an acute, hypoxia-like, pressor response in PASMCs  , which could interact in different ways with ion channels  . Using Pticulum) . In our channels . In addichannels .2 sensing by chemoreceptor glomus cells in the carotid body (CB), the prototypical acute O2 sensing organ in mammals. CB glomus cells are innervated by sensory fibers connected to brainstem centers that trigger a reflex hyperventilatory response and increased cardiac output in response to hypoxemia. In the MMS model, hypoxia produces a decrease in the activity of cytochrome c oxidase (CCO), thereby leading to a backlog of electrons along the ETC, with an increase in the reduced quinone (QH2)/quinone (Q) ratio and a slowdown of mitochondrial complex (MC) I. These changes result in the accumulation of NADH and production of a rapid and reversible increase in ROS at the mitochondrial intermembrane space (possibly H2O2 generated in MCI and MCIII). After equilibration with the cytosol, these signals modulate plasmalemmal ion channels to induce cell depolarization. Monitoring mitochondrial ROS production with the genetic probes described above for PASMC  model was proposed to explain acute Oor PASMC  showed tor PASMC .2 sensing was initially suggested given that mice deficient in NDUFS2, a core subunit essential for MCI assembly and NADH dehydrogenase activity (Ndufs2 expression with interfering RNA (siRNA) selectively blocked the contractile response of PASMCs to hypoxia without affecting responses to other vasoconstrictor agents , may alKCNK3 gene (coding TASK-1 channels) have been reported in patients with sporadic and familial pulmonary hypertension  channel activity in dispersed PASMCs and reported a contribution of these channels to HPV . Some stsion see .2-sensitive voltage-dependent K+ current in PASMCs has been proposed to be slowly inactivating, sensitive to 4-AP, and resistant to charybdotoxin. This profile would exclude several types of K+ channels  as HPV mediators   , is knowKd 1 μM) , that arKd 1 μM) . However2+-permeant cationic TRP channels, in particular TRPC1, TRPC6 and TRPV4, are highly expressed in resistance pulmonary myocytes where they contribute to Ca2+ entry and contraction   . The pre current .2+ channels in myocytes along the pulmonary arterial tree is strongly supported by the altered effects of low PO2 on cytosolic Ca2+ homeostasis in intact non-dialyzed VSM cells from the corresponding pulmonary arterial segments. As observed in other cells types, arterial myocytes (pulmonary and systemic) in vitro exhibit spontaneous oscillations of intracellular [Ca2+] (Ca2+ spikes), which largely represent the rapid release of Ca2+ from intracellular stores  expressed in the heart has specific cysteine residues whose oxidation could increase the probability of channel opening .The differential modulation by hypoxia of L-type Car stores . These Cnels see . The rembolition . In conte spikes . In moste spikes , hypoxiaikes  due to active Cl− transport, with the Cl− equilibrium potential (near 0 mV) in these cells being more positive than the resting potential (∼−50 to −60 mV)  Cl− currents. In contrast, increases in cytosolic [Ca2+] most frequently produce outward (hyperpolarizing) K+ currents in conduit myocytes  . Therefomyocytes   are potentiated by the activation of other ion channel classes to jointly produce a robust increase in cytosolic [Ca2+] and contraction.Taken together, the available experimental evidence suggests that the effector pathways mediating HPV are complex, as they do not depend on the modulation of a specific ion channel type. However, the level of expression of the main ion channel classes mediating HPV is higher in resistance myocytes than in conduit myocytes. It seems that, as occurs in CB chemoreceptor cells see , signali2 is a potent vasoactive agent and a major regulator of systemic vascular tone in vivo  channels and voltage-sensitive L-type Ca2+ channels. Membrane hyperpolarization caused by the activation of KATP channels or the inhibition of voltage-dependent Ca2+ channel opening can both lead to a decrease in Ca2+ influx and to VSM relaxation. However, these mechanisms may not be mutually exclusive, and interactions between them are highly probable   . In rat  hypoxia . Current2+ influx independently of KATP channel activation. As shown above  and species , where hypoxemia normally produces vasodilatation (2+ channel α1C subunit expressed in human embryonic kidney (HEK) cells in a manner indistinguishable from that observed in native smooth muscle L-type Ca2+ channels  expressed in mammalian cell lines are reduced in amplitude by lipophilic oxidizers of sulfhydryl groups, with these effects reverted by agents that reduce disulfide bonds  is required for the hypoxic modulation of channel function. It is likely that besides ROS, hypoxia generates other signals  that also contribute to the physiological regulation of L-type Ca2+ channel gating. In this respect, it is relevant to note that hemeoxygenase (HO), a broadly distributed enzyme that degrades heme and produces CO, was been suggested to directly modulate cardiac L-type Ca2+ channels by binding directly to the C-terminal end of the α1C subunit  which contain a specific and varied set of plasmalemmal ion channels that trigger extracellular Ca2+ influx and contraction during hypoxia. Hypoxia can also directly or indirectly induce Ca2+ release from intracellular stores. Whilst much progress has been made during the last decades in defining the molecular processes underlying HPV, uncertainty remains about the O2-sensing mechanism and the signals that modulate ion channel activity. While it appears that mitochondria act as O2 sensors in pulmonary resistance myocytes, it remains to be unequivocally demonstrated, however, that ROS and other mitochondrial signals are directly responsible for modulating membrane ion channels. Moreover, although important advances have been made recently, the molecular determinants of mitochondrial specialization in lung arterial myocytes, in particular those responsible for the apparent low O2 affinity of cytochrome c oxidase, remain to be fully clarified. In addition to its physiological value, HPV is of pathophysiological interest since, if maintained, it can lead to pulmonary hypertension. Given the medical importance of this pathology, it is important to continue to make progress in understanding how the initial (acute) activation of myocytes by hypoxia leads to the cellular proliferation and vascular remodeling phenomena characteristic of pulmonary hypertension.Acute O2 sensing apparatus in systemic myocytes and how the sensor signals to membrane ion channels are still to be defined. Existing data suggest that inhibition of L-type Ca2+ channels by hypoxia plays a key role in HVD, but further experimental data are needed to definitively clarify this signaling pathway. The molecular description of HVD may provide new pharmacological targets of interest in clinical conditions such as coronary and cerebral ischemia or arterial hypertension. This pharmacological development may acquire even greater medical relevance if it is taken into account that inhibition by hypoxia of Ca2+ channels in cardiac myocytes, which share the pore-forming α1C subunit with arterial myocytes, may have a cardioprotective role in the event of cardiac ischemia. The fields of HPV and HVD face relevant and highly attractive challenges that are important in fundamental research, but which also have clear translational implications in cardiocirculatory pathophysiology and pharmacology, which must be addressed in future experimental work.In contrast to resistance myocytes, myocytes in conduction pulmonary arteries relax with hypoxia; a response that is similar to the HVD seen in most systemic arteries. Although HVD has a fundamental physiological role, especially in the coronary and cerebral circulations, its molecular determinants remain poorly understood. The nature of the acute O


We developed a highly contiguous chromosome-level reference genome for North American bison to provide a platform to evaluate the conservation, ecological, evolutionary, and population genomics of this species. Generated from a F1 hybrid between a North American bison dam and a domestic cattle bull, completeness and contiguity exceed that of other published bison genome assemblies. To demonstrate the utility for genome-wide variant frequency estimation, we compiled a genomic variant database consisting of 3 true albino bison and 44 wild-type pelage color bison. Through the examination of genomic variants fixed in the albino cohort and absent in the controls, we identified a nonsynonymous single nucleotide polymorphism (SNP) mutation on chromosome 29 in exon 3 of the tyrosinase gene (c.1114C>T). A TaqMan SNP Genotyping Assay was developed to genotype this SNP in a total of 283 animals across 29 herds. This assay confirmed the absence of homozygous variants in all animals except 7 true albino bison included in this study. In addition, the only heterozygous animals identified were 2 wild-type pelage color dams of albino offspring. Therefore, we propose that this new high-quality bison genome assembly and incipient variant database provides a highly robust and informative resource for genomics investigations for this iconic North American species. Bison bison) are an iconic species due to their cultural and spiritual connection with Native American people, production potential to ranchers, and symbology of successful conservation efforts in federal and state parks and wildlife refuges. In addition, American bison were officially named the national mammal of the United States of America in 2016. Millions of bison once roamed the continent until they experienced a near extinction-level event reducing their census size by over 99% in the late 1800s (North American bison (te 1800s . Their sBos taurus) reference genome has primarily been used for bison whole genome analyses  × Brahman (Bos taurus indicus) cross and has since been used to generate other highly contiguous reference genomes  gene (c.64G>A) is associated with a dilution phenotype in Charolais cattle that causes a pale pelage color  have been shown to cause albinism in many mammalian species, including cattle (SLC45A2 gene (MITF (MLPH (MC1R (Identifying variant(s) associated with a white pelage color phenotype, specifically albinism, in bison provides facile phenotypic identification, cultural significance, and aesthetic value. Pelage color variation and albinism genetics have been studied extensively with many genes associated with a white pelage color phenotype in a multitude of mammalian species. A characterized point mutation in premelanosome protein 17  gene. Physiologically, the protein tyrosinase converts tyrosine to L-dihydroxyphenylalanine (DOPA) which is converted to dopaquinone, the latter of which is converted to melanin  “Molly” (SAMN33913325), a male domestic cattle angus-cross (B. taurus) herd representative (SAMN33913326), and a female F1 bison-cattle offspring “Midnight” from a private ranch in Texas (SAMN33913332). Biopsy tissue samples were collected from the bison dam and domestic cattle bull. Fresh blood and sterile ear punch samples were collected from the F1 bison-cattle hybrid. A cell line was established from the F1 hybrid and karyotyped by the Molecular Cytogenetics Laboratory at Texas A&M University School of Veterinary Medicine to confirm species identification . Metaphase cells were harvested with demecolcine solution , treated with Optimal Hypotonic Solution (Rainbow Scientific), and fixed in 3:1 methanol/acetic acid. The cells were dropped on clean, wet glass slides and checked under phase contrast microscope (×200) for quality. Chromosomes were stained by GTG-banding , using the Nanobind CBB Big DNA Kit  to yield high molecule length DNA. Four single molecule real-time (SMRT) cells were run in Circular Consensus Sequence (CCS or Hifi) mode and generated a total average sequence depth of 120×, given an approximately 3 Gb genome.DNA was extracted from the tissue, hair, and blood samples using the DNeasy or Gentra Puregene kit (Qiagen) according to manufacturer protocol . A totalB. bison) “Molly”, Angus-cross domestic cattle bull (B. taurus), and female F1 hybrid “Midnight” (The F1 hybrid Hifi sequence reads were sorted into haplotype contigs with Canu v2.1.1 using the trio binning assembly approach (idnight” . The bisChromatin conformation capture (Hi-C) data was previously generated by DNA-Zoo and downloaded from NCBI, accession SRR8616930 . ContigsNext, Bionano optical mapping was used to further improve scaffolds. Optical mapping data was generated and analyzed according to the established Saphyr workflow (Bionano Genomics). Briefly, high molecular weight DNA was extracted from a female bison fibroblast cell line according to Bionano Genomics: Bionano Prep Cell Culture DNA Isolation Protocol. Once the DNA concentration was even throughout the solution and within the target parameters, it was labeled with Direct Label Enzyme (DLE-1) as stated in the Bionano Prep Direct Label and Stain (DLS) Protocol. The generated Bionano data was used assemble the haplotype with the options set to keep conflicts for both the Bionano and sequence assemblies and trimming overlapping sequence contigs turned off.Bos taurus ARS-UCD1.2 using MUMmer4's nucmer4 (https://dot.sandbox.bio). The ARS-UCD1.2 (Bos taurus) chromosomes were then aligned to each corresponding Bison scaffolds using D-Genies, minimap2 option, to orient and order the bison scaffolds into chromosomes . The transition from cytosine to thymine in the first position of the codon causes an amino acid substitution of glycine for arginine at the protein position 372  in 7 albino bison, shown in the heterozygous state in the dams, and absent in 317 bison with a wild-type pelage color is suggestive of this SNP being the causative mutation in these animals. These results agree with an autosomal recessive inheritance of oculocutaneous albinism type 1 with the known dams shown as carriers for albinism, similar to what is seen in humans (The observation of a mutation identified in exon 3 of the Brucella abortus in Yellowstone National Park (With the discovery of an albinism-associated mutation, we have provided a framework to use this genomic resource to uncover the genetic bases for important phenotypes in bison. Bison are a valuable species as both wildlife and livestock and therefore, many groups benefit by further understanding bison genomics. One such relative issue is immune response. Since bison and domestic cattle have been in proximity since domestic cattle's arrival to North America, many diseases are transmitted between the 2 species. However, in some cases, these species are known to react different to both pathogens and vaccines (This high-quality annotated bison genome will also facilitate multiple arenas of future bison genetic research. For example, bison specific whole genome approaches will be possible without the potential biases inherent when using the cattle reference genome. In addition, population genetics studies will no longer need to rely on limited microsatellites  or soleljkad156_Supplementary_DataClick here for additional data file.


Self-renewal and differentiation of skeletal stem and progenitor cells (SSPCs) are tightly regulated processes, with SSPC dysregulation leading to progressive bone disease. While the application of single-cell RNA sequencing (scRNAseq) to the bone field has led to major advancements in our understanding of SSPC heterogeneity, stem cells are tightly regulated by their neighboring cells which comprise the bone marrow niche. However, unbiased interrogation of these cells at the transcriptional level within their native niche environment has been challenging. Here, we combined spatial transcriptomics and scRNAseq using a predictive modeling pipeline derived from multiple deconvolution packages in adult mouse femurs to provide an endogenous, in vivo context of SSPCs within the niche. This combined approach localized SSPC subtypes to specific regions of the bone and identified cellular components and signaling networks utilized within the niche. Furthermore, the use of spatial transcriptomics allowed us to identify spatially restricted activation of metabolic and major morphogenetic signaling gradients derived from the vasculature and bone surfaces that establish microdomains within the marrow cavity. Overall, we demonstrate, for the first time, the feasibility of applying spatial transcriptomics to fully mineralized tissue and present a combined spatial and single-cell transcriptomic approach to define the cellular components of the stem cell niche, identify cell‒cell communication, and ultimately gain a comprehensive understanding of local and global SSPC regulatory networks within calcified tissue. Disruption of SSPC regulation is typically caused by localized changes within the bone marrow niche.3 Thus, identifying and characterizing the SSPCs within their local regulatory environment is an area of intense research.In healthy bone, skeletal stem and progenitor cells (SSPCs) are tightly regulated to self-renew, to maintain the stem cell pool, and to differentiate to replenish the population of bone-forming osteoblasts.7 A variety of markers have been proposed using transgenic mice and immunohistochemistry to label SSPC populations, including platelet-derived growth factor receptor alpha (PDGFRα) and SCA1 (termed PαS cells),8 leptin receptor (LEPR), and CXCL12 .10 The application of scRNAseq has significantly advanced our understanding of the intrinsic signaling mechanisms in these SSPC populations.12 However, single-cell studies have revealed that previously proposed markers label discrete stromal cell subtypes, supporting the concept of heterogeneity within the proposed SSPC populations. These studies have also shown that previously identified stromal cells have expression profiles that overlap with markers for committed adipocyte lineage cells, such as Adipoq, and that these SSPC markers may also label progenitor cells already primed to the osteogenic and adipogenic lineages.7 This lack of consensus highlights the complexity of stem cell biology within the bone and underscores the need to better understand the mechanisms driving this diversity.While single-cell RNA sequencing (scRNAseq) has become widely used and can interrogate each individual cell within a tissue, the requirement for dissociation, often through enzymatic digestion, not only alters the transcriptional profile of each cell but also removes the ability to place these cells within their native environment. The ability to interrogate the spatial profile of these cells relative to each other in tissue only recently came about with the development of the spatial transcriptomics platform. While this technique is easily applied to soft tissue, the need to decalcify bone, a process that typically destroys mRNA, has remained an obstacle for long bone samples. As a result, scRNAseq has been the primary approach to dissect SSPC populations in long bone.14 However, understanding these spatial relationships in vivo in an unbiased manner has proven challenging. Researchers have applied large-scale scRNAseq to multiple cell types in an attempt to computationally reconstruct the stem cell niche and assign primary regulatory factors to their key cell types of origin.12 However, in addition to lacking any spatial information, this technique is limited in disentangling cell‒cell communication due to numerous secreted factors that are derived from the diverse cell types present within the bone marrow environment. As such, a more comprehensive understanding of the in situ SSPC niche microenvironment that places these cells in the spatial context of major niche modulators, such as the bone surface or blood vessels, is needed.4The finding that SSPC populations have multiple transcriptional profiles may be due to various factors. One possibility is that their transcriptional profile can be dictated by their localization within the marrow, a spatial interrogation difficult to assess by scRNAseq. Stem cells are regulated by the secretion of soluble factors by neighboring cells that comprise the stem cell niche. Subtle shifts in the spatial location or changes in the cell‒cell interactions of SSPCs and niche-forming marrow resident cells can have a profound impact on SSPC fate and function.16 or to unravel heterogeneity and spatial organization in tumors,18 this technique has only recently begun to be utilized in the context of musculoskeletal tissue. Early studies used spatial transcriptomics in soft tissue such as tendon20 or muscle22 and, most recently, we utilized this technique for mineralized bone tissue such as the developing calvarial suture23 or regenerating digit tip.24 Barring the need for tissue decalcification while still preserving mRNA in long bones, limitations in spatial resolution remain a major drawback given that each spatial spot within the Visium spatial gene expression system from 10 × Genomics represents 55 µm. Thus, a single spatial spot can encompass numerous diverse cell types in tissues with high degrees of cellularity and heterogeneity, making it challenging to determine whether changes in gene expression are the result of changes in cell composition within each spot, changes in gene expression within the cells comprising each spatial spot, or a combination of the two. In the context of intact musculoskeletal tissue, this limitation can be resolved (1) if the tissue area represents relatively low cellularity in which each spatial spot would correspond to only a few cells or (2) if cells of similar origin and transcriptome are present within each spot. However, with more heterogeneous tissue, in which cell types of very different lineages coexist within a tightly confined space, these spots can encompass tens of cells with highly unique transcriptional profiles, making it difficult to ascertain the cause of observed transcriptional changes. The bone marrow stem cell niche is a prime example of this type of composition, where cells derived from vascular, hematopoietic, and mesenchymal lineages exist within tight spatial confines, carefully regulating each other through direct and indirect signaling mechanisms.14 Taken together, the technical difficulties and tissue composition of the bone marrow niche have made it a difficult target for spatial transcriptomics.Spatial transcriptomics bypasses the need to generate single-cell suspensions, preventing the introduction of potential artifacts by enzymatic digestion and providing unbiased transcriptional profiling in vivo. While widely used on soft tissues such as the brain7 Using correlative analyses, we then mapped out cellular subtypes enriched within these SSPC-containing spatial spots, with Pdgfra+Sca1+ SSPCs preferentially localizing to the periosteal surface and Cxcl12+Lepr+ SSPCs enriching within the marrow. Finally, we used cell‒cell interaction analyses, spatial gene expression, and spatial-time analyses24 to reveal signaling networks utilized within the niche and across microdomains within the bone. This study overcomes technical and analytical obstacles for spatial transcriptomics in long bone to assess, for the first time, changes in SSPC regulation within its in vivo context.To overcome the spatial limitations outlined above, we combined spatial transcriptomic data with bone marrow scRNAseq and several different predictive modeling packages to deconvolve the larger spatial spots into their cellular constituents. Using this approach, we first spatially localized SSPC subtype populations previously identified using scRNAseq.Col1a1, Bglap), with additional periosteal (Postn) and osteoprogenitor  genes enriched within the cortical and trabecular bone, respectively. In contrast, hematopoietic , erythropoietic (Hba-a2), and proliferative markers were heavily enriched within the bone marrow  (PαS), and CXCL12-abundant reticular (CAR) cells, demarcated here by the expression of Cxcl12 and Lepr. To characterize the spatial distribution of these SSPC clusters, we applied a similar predictive modeling workflow as described above, with MesLin+ spatial spots further divided into these two proposed SSPC cell subtypes, along with mature osteoblasts to serve as a control , while both Mac1 and Mac4 associations were found to not be significant  or Cxcl12 and Lepr (CAR). Using our predictive pipeline, we exploited the strengths of each analytical approach to create a predictive workflow for the location of each cell cluster within complex and heterogeneous spatial data. Our spatial predictive mapping suggests that CAR cells are heavily enriched within the bone marrow, consistent with previously published results.34 In contrast, while some PαS cells were found within the bone marrow, these cells were heavily enriched within the cortical tissue primarily along the outer periosteal surface. This finding is consistent with more recent studies identifying PDGFRα and SCA1 as markers of periosteal stem cells important for fracture healing.37 These findings suggest that either PαS cells present within the bone marrow show significant overlap in gene expression with stem cells within the outer periosteum or that periosteal cells may have been inadvertently captured during enzymatic digestion to generate bone marrow single-cell preparations.While scRNAseq analyses have substantially advanced our understanding of stromal cells and their commitment to cell-specific lineages within the bone, some disparity remains in terms of the heterogeneity of proposed stromal cell populations. Prior immunohistochemical and in situ studies have identified several SSPC markers, including PDGFRα/SCA1,12 as well as in regulating SSPC proliferation and differentiation.40 Conversely, other studies have shown that mesenchymal stromal cells induce M2 polarization within macrophages coinciding with elevated levels of Jak/Stat signaling.42 Notably, due to the spatial resolution (55 µm), our signaling analyses cannot distinguish cells in immediate contact with SSPCs from those present within the same spatial spot but still upward of several cell lengths removed from the SSPC itself. While this method showed improvements compared to analyses relying on scRNAseq alone, pathways such as Notch, which require direct cell‒cell interaction, may still be falsely predicted. Combining spatial transcriptomics, scRNAseq, and our spatial deconvolution pipeline, our results demonstrate the ability to dissect cellular composition and communication within the niche in an unbiased fashion.SSPCs rely on the regulatory function of other cell types within the stem cell niche to be maintained in an undifferentiated state. To date, several cell‒cell communication prediction packages have been developed to infer ligand‒receptor-based interactions. However, one striking limitation of these methods remains the lack of spatial information. As a result, while these communication packages can predict potential mechanisms through which crosstalk may occur, they lack the ability to determine if the proposed crosstalk, while theoretically possible, is biologically feasible . To overcome this limitation, we made use of the fact that spatial transcriptomics is not at a single-cell resolution and instead considered each spatial spot at the “niche” level. We determined the probability that other cell types of the bone marrow were present within the same spatial spot as our previously identified SSPC populations. Consistent with previously published results, our data indicate that smooth muscle cells, endothelial cells, macrophages, and, to a lesser extent, megakaryocytes comprise the SSPC niche. Reanalysis of both endothelial cells and macrophages identified cellular subtypes enriched within the niche. Transcriptional analyses of the endothelial and macrophage subtypes present within the niche revealed enriched activation of Notch and Jak/Stat signaling, respectively. Previous studies have shown the critical role of endothelial Notch signaling in regulating hematopoiesis,43 smooth muscle cells,44 and skeletal stem cells45 all favor glycolysis as an energy production mechanism to minimize ROS production46 and overall gene activation.48 Additionally, these results showed elevated levels of transcripts associated with FA metabolism near the cortical bone surface. This finding is consistent with in vitro studies that show increased FA metabolism in mature osteoblasts49 and indicate the importance of FA metabolism in normal bone accrual50 and the response to anabolic stimuli.51 Similarly, previous findings of elevated WNT53 and PDGF55 signaling near vessels as well as high activation of PDGF56 and BMP58 near bone surfaces were reflected in this analysis. Our findings further add indications of the potential range of these coordinate gradients, with, for example, WNT signaling showing a more restrictive window of activation surrounding blood vessels than that observed by PDGF signaling.While the niche has been extensively investigated, one concept that is well known, yet difficult to assess, is the formation of microdomains within the bone marrow regulated by the release of growth factors from various tissues. Osteoblasts and osteoclasts along bone surfaces, chondrocytes at the terminus of the growth plate, and vascular endothelial and smooth muscle cells have all been shown to regulate the local environment. However, assessing these signaling gradients in an unbiased fashion has proven difficult with existing techniques. Our analyses revealed unique expression of transcripts related to metabolism and major morphogenetic pathways. Despite the presumably high availability of oxygen near blood vessels, we observed low levels of oxidative phosphorylation and high levels of glycolysis within spots immediately adjacent to vascular tissue. These results are consistent with previous data that suggest that endothelial cells,Several potential limitations should be considered in this analytical pipeline. The unique anatomy of bone, with a mineralized outer cortical shell, can delay the diffusion of reagents to the internal marrow and necessitate decalcification, ultimately delaying sample processing. These factors may contribute to the relatively low number of transcripts retrieved compared to those of previous works in soft tissue. While previous studies using scRNAseq of bone-derived cells or spatial transcriptomics of soft tissue typically yield several thousand transcripts per cell/spot, our results show a greatly reduced efficiency. As such, limitations in the extent and types of analyses possible should be carefully considered. These limitations were overcome, to some extent, by (i) more broadly selecting our cell clusters of interest for spatial mapping, (ii) investigating expression patterns of groups of genes linked to a common pathway, (iii) relying on a combination of predictive packages, and (iv) using techniques such as SpatialTime to allow the merging of multiple samples into a single analysis. Optimization of this sample preparation pipeline may yield greater transcript recovery, increasing the accuracy of downstream analyses and permitting more sophisticated investigations in the future. While we speculate that our findings will be similar across mouse strains, our spatial analyses are specific to C57BL/6 mice. Additional studies are needed to determine global versus strain-specific findings.Overall, this study makes use of predictive modeling, combining the unbiased transcriptional profile and in vivo spatial context of spatial transcriptomics with the cellular resolution of scRNAseq. These studies reveal the cellular and signaling components of the stem cell niche, as well as the gradients of signals established by various anatomical structures within the bone. These analyses are highly supportive of intensive studies published over the past several decades and suggest that this analytical pipeline could serve as a method to unbiasedly assess changes in several aspects of stem cell and bone biology in response to perturbation or therapeutic intervention.−1pH 8 EDTA for 2 weeks on a shaker, with a fresh reagent change every 1–2 days. The samples were then processed for paraffin embedding. All processing was conducted with minimal delay between steps. Two 5 µm longitudinal sections through the marrow were collected per block from two different FFPE blocks  and placed onto the Visium Spatial Gene Expression Slide (10 x Genomics). Spatial transcriptomic libraries were generated using the Visium Spatial Gene Expression for FFPE Kit according to the manufacturer’s instructions . Briefly, the slide was subjected to H&E staining and imaged at × 20 magnification using a NanoZoomer S60v2MD (Hamamatsu). Tissue sections were decrosslinked and probe-hybridized (Visium Mouse Transcriptome Probe Set v1.0), targeting 20 551 mouse genes. Specifically, hybridized and ligated probes were then released from the tissue through permeabilization and captured by Visium Slide oligos. Barcoded ligation products were then subjected to amplification and indexing. Libraries were pooled and subjected to 2 × 96 bp pair-ended sequencing with a sequencing depth of >100 M reads per sample on a NovaSeq instrument (Illumina). Demultiplexing and sequence alignment, along with registration to H&E-stained images, was carried out using the SpaceRanger pipeline.Femurs from 2 separate mice were harvested from 10-week-old C57BL/6 mice (Charles River), bisected, and immediately fixed in 10% buffered formalin overnight at 4 °C. The samples were decalcified in 0.5 mol·LR package Seurat.29 For spatial data, histologically unique regions of the bone were manually segmented, and differential gene expression was carried out using default parameters. For whole bone marrow single-cell analyses, datasets from GSE145477 (GSM4318799)7 and GSE128423 5 were merged into a single object to represent all major types of cells present within the bone marrow. For analysis of mesenchymal lineage cells, datasets from GSE145477 7 were reanalyzed. Pathway analysis of DEGs was conducted using the Database for Annotation, Visualization and Integrated Discovery (DAVID).59 The AddModuleScore function was used to assess levels of overall pathway activation using established gene lists curated from KEGG pathways  and 1 (most distant from surface). For analysis of genes differentially expressed across each of these spatial-time axes, marrow spatial spots were analyzed by the R package Monocle using the differentialGeneTest function, replacing pseudotime values with self-calculated spatial-time values. For visualization of changes in cellular pathways, module scores were calculated across SpatialTime values, and a smoothed curve was generated by averaging neighboring spot values.SpatialTime analyses were conducted according to previously published methods.For immunofluorescence, long bones from 12-week-old mice were isolated and fixed in 4% PFA overnight. Bones were then decalcified in Decalcifier I  for 4 days. Bones were cryoprotected in 20% sucrose and 2% polyvinyl pyrrolidine (PVP) and then embedded in 20% sucrose, 2% PVP, and 8% gelatin. Sections were cut at a thickness of 100 µm and stained using primary antibodies against endomucin , PDGFRα , or p-SMAD3 , followed by secondary antibodies . Images were acquired using a Leica DMI8 confocal microscope. Images are representative of three individual samples taken from three separate mice.Mus musculus duplex probes Ppib, green channel, and Polr2a, red channel, and the single channel Ubc control to test for high expression in bone) and one negative control probe (Escherichia coli DapB) were used. Following protocol optimization, sections were assessed for Lepr (C1) and either Cd44 (C2), Cd74 (C2), or Ryr1 (C2). In situ hybridization was performed according to the protocol of the RNAscope 2.5 Duplex Detection Kit . Briefly, sections from FFPE mouse tibias and an RNAscope Control Slide  were baked at 60 °C for 1 h before deparaffinization and dehydration according to the protocol. The slides were then incubated with H2O2 and permeabilized before air drying. C2 probes were diluted in C1 probes at a 1:50 ratio and incubated on the slides for 2 h at 40 °C and amplified according to the protocol. Positive Ppib/Polr2a duplex probes, DapB negative probe, and Ubc control probe were incubated separately on three individual tibia samples. The RNAscope Control slide received the duplex control probes. Prior to coverslipping, slides were stained with hematoxylin. Images are representative of three individual samples. For quantification of RNAscope, 50 Cd44+, Cd74+, and Ryr1+ cells were identified, and the distance to the nearest Lepr+ cell was manually quantified using NDP.view2 (Hamamatsu).To detect single mRNA molecules, we performed FFPE-fixed RNAscope on sections from control C57BL/6 mice (Charles River). Positive control probes (the Figure S1Figure S2Figure S3Table S1Table S2Table S3Table S4Table S5Table S6Table S7Supplementary figure legends