Ketamine, a multimodal anesthetic drug, has become increasingly popular in the treatment of pain following traumatic injury as well as treatment-resistant major depressive disorders. However, the psychological impact of this dissociative medication on the development of stress-related disorders such as post-traumatic stress disorder (PTSD) remains controversial. To address these concerns, preclinical studies have investigated the effects of ketamine administration on fear memory and stress-related behaviors in laboratory animals. Despite a well-documented line of research examining the effects of ketamine on fear memory, there is a lack of literature reviews on this important topic. Therefore, this review article summarizes the current preclinical literature on ketamine and fear memory with a particular emphasis on the route, dose, and timing of ketamine administration in rodent fear conditioning studies. Additionally, this review describes the molecular mechanisms by which ketamine may impact fear memory and stress-related behaviors. Overall, findings from previous studies are inconsistent in that fear memory may be increased, decreased, or unaltered following ketamine administration in rodents. These conflicting results can be explained by factors such as the route, dose, and timing of ketamine administration; the interaction between ketamine and stress; and individual variability in the rodent response to ketamine. This review also recommends that future preclinical studies utilize a clinically relevant route of administration and account for biological sex differences to improve translation between preclinical and clinical investigations. Ketamine has become a multi-purpose tool within the field of medicine for a variety of indications from trauma analgesia to a treatment for depression and post-traumatic stress disorder (PTSD). Due to the preservation of ventilation and favorable hemodynamic stability, ketamine has gained popularity among military and civilian first responders as the primary analgesic medication of choice to treat pain caused by traumatic injury  and is tDue to the inherent moral dilemmas of conducting prospective clinical experiments involving traumatic events and stress-related disorders in humans, researchers often turn to preclinical rodent models to explore ketamine’s effects on fear memory and PTSD-like behaviors. However, results from preclinical research are often contradictory due to a variety of confounders. Despite a well-documented line of research examining ketamine’s effects on rodent fear memory, there is a lack of published reviews on this topic. Therefore, this review aims to describe the most recent preclinical literature that has explored the effects of peri-trauma ketamine administration on rodent fear memory and PTSD-like behaviors with consideration of the route, dose, and timing of ketamine administration along with additional variables that may affect ketamine effects on fear memory. Furthermore, this review summarizes the potential biological mechanisms by which ketamine may interfere with fear memory in rodents and suggests recommendations for future preclinical studies that will enhance clinical translation.Rodent fear memory is comprised of multiple phases including acquisition, extinction, and recall. Fear memory acquisition can occur through various methods, but the most common is through Pavlovian fear conditioning, where a neutral stimulus (sound tone) is paired with an aversive stimulus (footshock) such that a future exposure to the neutral stimulus produces freezing behavior. Humans undergo the same phenomenon in response to a traumatic event is often associated with sights, sounds, or smells present at the time of trauma. Fear extinction refers to a process whereby rodents slowly reduce freezing behaviors when repeatedly exposed to a non-reinforced neutral stimulus (sound tone with no footshock). Essentially, a rodent is creating a new, safe memory that overwrites the previous fear memory. The concept of fear extinction underlies the process of cognitive behavioral therapy in humans, whereby a patient will describe or discuss a traumatic event in repeated sessions. Lastly, fear recall occurs after a rodent has successfully demonstrated fear extinction and is re-exposed to the original environment where fear memory acquisition took place. The neutral stimulus and/or the original fear conditioning environment alone can trigger freezing behavior. Similarly, humans can experience this same phenomenon when visiting the scene of a previous traumatic event or experiencing a sight, sound, or smell previously associated with the trauma.R,S) ketamine doses; (3) the utilization of a rodent fear conditioning paradigm; (4) ketamine’s effects on fear memory acquisition, fear memory extinction, and fear recall; and (5) male or female sex. We utilized the following exclusion criteria: (1) human or non-rodent studies; (2) anesthetic ketamine doses; and (3) chronic ketamine administration. We included studies only with subanesthetic ketamine doses because these are clinically relevant doses that are recommended for trauma analgesia without severe dissociation [For this review, we utilized the following inclusion criteria: (1) rodent studies including mice or rat species; 2) subanesthetic racemic  route to administer ketamine to rodents, despite the intravenous (IV) route being the preferred route of ketamine administration in patients. The differences in route of administration can lead to conflicting results, as evidenced by IP and IV ketamine producing opposite effects within the same experimental paradigm. A 10 mg/kg IP injection of ketamine facilitated the extinction of fear memory, while a 10 mg/kg (2 h) IV infusion delayed fear extinction in rats . These rIn addition to the route of administration, the dose of ketamine administered also plays a crucial role in the differential effects of ketamine observed on fear memory. Many studies have reported dose-dependent effects of ketamine on fear memory. A dose of ketamine at 10 mg/kg is frequently observed within the preclinical literature and is often cited as an effective antidepressant dose in rodents . In ratsResearchers utilize the timing of ketamine administration in fear conditioning studies to simulate different treatment models. For example, administering ketamine before fear conditioning represents a prophylactic treatment; administration immediately after fear conditioning mimics trauma analgesia; and a delayed dose after fear conditioning or prior to extinction training simulates a therapeutic modality. Ketamine exerts differential effects depending on when it is administered in relation to the exposure to fear conditioning or extinction training—in other words, timing matters. The sections below include a review of preclinical results from ketamine administered before fear conditioning, immediately after fear conditioning, and at later time points after fear conditioning (including administration before fear extinction testing).There are a limited number of studies that have examined the effects of ketamine administered prior to fear conditioning on fear memory behaviors. These types of models are generally referred to as prophylactic in that the researchers evaluate the protective effects of ketamine on fear memory if administered prior to a trauma. Most studies vary with regards to the ketamine dose  and the amount of time between ketamine administration and fear conditioning (30 min to 1 month), along with the time until the fear memory assessments (1 to 11 days). For example, ketamine administration  15 min before contextual fear conditioning in rats decreased fear memory when measured the next day . CliftonThe effects of subanesthetic ketamine doses administered in the immediate period following fear conditioning on short-term freezing behavior are mixed. Generally, the premise is that post-trauma ketamine may serve to either enhance or disrupt memory consolidation and thus impact fearful memory formation and the development of stress-related disorders. A pair of investigations administered ketamine at a higher dose range  immediately following foot-shock fear conditioning and found no effects on freezing behavior when it was measured 24–48 h later ,17. In cIn addition to the effects on short-term memory, several investigations have tested the effects of ketamine administered immediately after fear conditioning on long-term fear memory. For instance, Kulyk et al. 2017) administered ketamine  immediately after fear conditioning and found reduced freezing during initial extinction learning 10 days later, but no overall effect on freezing behavior during fear extinction recall testing 11 days later in rats . Similar17 adminiPreclinical researchers also delay ketamine administration after fear conditioning to mimic the use of ketamine as a pharmacological treatment for stress-related disorders. Delayed ketamine treatment models typically occur at 24 h post-fear conditioning or in the immediate period (30–60 min) prior to fear extinction testing. Girgenti et al. 2017) reported decreased freezing on the second day of extinction testing following low-dose ketamine  administration 24 h after fear conditioning in rats . This su reportedIn addition to delayed ketamine models, researchers have administered ketamine prior to fear extinction testing to test the potential therapeutic effects on stress-related disorders such as PTSD. Higher ketamine doses  administered 30–60 min prior to fear extinction testing had no effects on freezing behaviors when they were tested at 2 days  after feThe experience of stress plays a crucial role in the development of psychiatric disorders, and is one of the diagnostic criteria for PTSD. Indeed, disruption of fear extinction can occur when rodents are exposed to various types of stressors such as the forced swim test, forced restraint, and maternal separation ,27,28. HIndividual variability is an important factor in determining the susceptibility or resilience to the development of certain stress-related disorders, as not all individuals exposed to a traumatic event develop PTSD. Consequently, researchers can expect individual differences among animals, and if correctly identified, these factors may predict individual susceptibility or resilience to PTSD. For example, a ketamine infusion  immediately after cued fear conditioning did not enhance long-term fear memory when rats were tested 1 week later . HoweverN-methyl-d-aspartate (NMDA) receptors but is relatively non-selective and, as such, modulates an array of other molecular systems [An increasing number of preclinical investigations aim to describe the molecular systems involved in the effects of ketamine on fear memory, as shown in  systems ,31. ThosN-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are two glutamatergic receptors that are central to learning, memory, and plasticity [The asticity ,33, withasticity ,35 and iasticity . A propoasticity ,37. Ketaasticity ; similarasticity . This reasticity . The rouasticity . Ketaminasticity . Furtherasticity . Althougasticity . By and BDNF is a protein associated with learning and memory that is often implicated in the antidepressant effects of ketamine ,38. In kAnother mechanism of ketamine’s action includes the mTOR and ERK pathways. The extracellular signal-regulated kinase (ERK) and the protein kinase B (AKT) pathways are upstream modulators of the mTOR pathway ,42 and a+-positive cells were increased in the hippocampus as a result of prophylactic ketamine  administered one week prior to contextual fear conditioning in mice, coinciding with reduced fear memory [Through interaction with the aforementioned molecular pathways, ketamine also exerts influence on many transcription factors such as c-Fos, ΔFosB, and Egr1. A descriptive study reported dose-dependent increases in c-Fos levels in the mPFC and amygdala, which are important limbic regions associated with fear memory, in rats following IV ketamine infusion  [r memory . c-Fos lr memory . ΔFosB, r memory . Prophylr memory . Early gr memory . A studyr memory . Taken t18F-FDG-MicroPET imaging two days after ketamine administration  in fear-conditioned rats revealed no change in glucose metabolism in the hippocampus, frontal cortex, or amygdala [18F-FDG-PET scan taken immediately after an IV ketamine infusion  revealed robust effects on regional glucose metabolism in rats [There is a lack of in vivo brain imaging studies using ketamine administration in rodents. Brain imaging, such as fluorodeoxyglucose-positron emission tomography (FDG-PET), can reveal regional differences in brain energy utilization in living animals. For instance, amygdala . This la in rats . The ket in rats . One of Stress hormones released following trauma regulate limbic activity and may enhance or impair memory formation based on their concentrations and durations of exposure within the brain ,50,51,52In summary, preclinical research on ketamine’s effects on fear memory has demonstrated inconsistent findings, with results showing increased, decreased, or no effect on fear memory in laboratory animals. However, there are multiple factors contributing to these differences, such as the inconsistent routes, doses, and timing of ketamine administration as well as stress exposure at the time of ketamine administration. Multiple brain regions and molecular pathways are implicated in the effects of ketamine on fear memory, although the findings in molecular systems are again dependent upon the route (bolus vs. infusion) and timing (pre- vs. post-fear conditioning) of ketamine administration in animals. Taken together, the route and the timing of ketamine administration are two variables that greatly impact the fear memory outcomes found within this review, and investigators should consider them when evaluating preclinical studies reporting ketamine’s effects on fear memory. With careful considerations of preclinical methodology, we can enhance our understanding of the behavioral and molecular mechanisms of ketamine’s effects on fear and stress-related disorders and improve clinical translation from the bench to bedside.R,S)-ketamine  and its metabolites -hydroxynorketamine (HNK) and -HNK  one week prior to contextual fear conditioning in mice; racemic ketamine and -HNK attenuated learned fear in male mice but not in female mice [Research teams have explored many variables within a variety of fear memory and fear extinction ketamine studies; however, the effects of sex and route of drug administration are often neglected. Male rodents were predominantly used in the ketamine preclinical literature for this review , which lale mice . Since bale mice . Therefo


Ionizing radiation is a critical aspect of current cancer therapy. While classically mature bone was thought to be relatively radio-resistant, more recent data have shown this to not be the case. Radiation therapy (RT)-induced bone loss leading to fracture is a source of substantial morbidity. The mechanisms of RT likely involve multiple pathways, including changes in angiogenesis and bone vasculature, osteoblast damage/suppression, and increased osteoclast activity. The majority of bone loss appears to occur rapidly after exposure to ionizing RT, with significant changes in cortical thickness being detectable on computed tomography (CT) within three to four months. Additionally, there is a dose–response relationship. Cortical thinning is especially notable in areas of bone that receive >40 gray (Gy). Methods to mitigate toxicity due to RT-induced bone loss is an area of active investigation. There is an accruing clinical trial investigating the use of risderonate, a bisphosphonate, to prevent rib bone loss in patients undergoing lung stereotactic body radiation therapy (SBRT). Additionally, several other promising therapeutic/preventative approaches are being explored in preclinical studies, including parathyroid hormone (PTH), amifostine, and mechanical loading of irradiated bones. Approximately half of the 1.7 million new annual cancer diagnoses will be treated with ionizing radiation therapy (RT). As improvements in cancer screening and treatments continue to decrease cancer mortality, patients are now living long enough to experience treatment-related toxicities that may have previously been obscured by cancer-related death. Mature bone has classically been thought of as radio-resistant tissue; however, recent pre-clinical and clinical studies have demonstrated that RT-induced bone loss and fracture are common problems with serious implications for patient quality of life (QOL) ,3.Stereotactic body radiation therapy (SBRT), for example, when used in the treatment of lung tumors near the chest wall, unavoidably delivers high doses of radiation to the ribs or vertebrae, and it can predispose patients to significant risks of radiation-induced rib fractures (RIRF), vertebral fractures, and chest wall pain (CWP) ,5,6. TheAs SBRT becomes a new standard approach for treating tumors throughout the body in the oligometastatic setting, the incidence of RT-induced bone toxicity is forecasted to increase over time ,17,18,19In women treated for endometrial adenocarcinoma with standard doses of RT per fraction (1.8–2 Gy per fraction), the 5-year pelvic fracture rate is ~30% ,21. LikeThe exact mechanisms of RT-induced bone damage are unclear. Utilizing clinical data alone to determine the effects of RT on bone is complicated by numerous confounding factors, including medical comorbidities such as osteoporosis/osteopenia or concurrent medications that may impact bone turnover/structure, such as chemotherapy or steroids. Therefore, research with animal models has been critical in identifying several mechanisms by which RT-induced bone injury may occur, as well as in identifying potential therapeutic approaches. Below we will: (i) review the preclinical studies that helped elucidate the relationship between irradiation and bone injury, (ii) focus on how these data have been applied to develop approaches for addressing clinical research questions related to radiation-induced bone loss, and finally, (iii) review how the most recent rodent studies have helped generate multiple therapeutic approaches.Numerous studies have demonstrated that skeletally mature humans who are exposed to RT experience higher incidence of bone fractures, decreased bone strength, and osteoporosis ,20. The These confounding factors often cloud the true impact of irradiation, but most of these factors are difficult to control in a clinical setting. Human data on irradiation of normal bone without the impact of weight bearing is mostly possible through research involving low earth orbit astronauts exposed to cosmic irradiation. Controlled preclinical studies however, utilizing murine and nonhuman primate models, have greatly advanced our understanding of the effects that irradiation has on the bone microenvironment both before and after skeletal maturity. As we gain more insight into the precise mechanisms of damage, we can begin to formulate preventative strategies for those at the highest risk of meaningful bone toxicities.Historically, the mechanisms of RT-induced bone damage were thought to be related to vascular injury as well as persistent osteoblast damage/suppression ,34. DamaAn acute, transient increase in the number and activity of osteoclasts is a putative mechanism for causing the pathologic fractures in bone that are caused by radiation therapy . TherefoThe cause for an early and transient increase in osteoclast activity is unclear, and perhaps due to signals between other cells, including osteocytes. Bone is made up of approximately 90–95% osteocytes . These cThis increase in osteoclast differentiation can be demonstrated by isolating marrow from irradiated bone marrow and cultured on a proxy bone surface. To further investigate this finding and directly measure osteoclast activity, hematopoietic cells from the femurs of C57BL/6 mice can be isolated, and then plated on a hydroxyapatite (HA) proxy for bone in 12-well plates using RANKL and macrophage colony-stimulating factor (MCSF) to promote osteoclast differentiation. By fourteen days after a 2 Gy X-ray dose, erosion of the HA surface area is increased by 390% relative to control . 45Ca kinetics late after exposing BALB/c mice to a single 16 Gy fraction that delivered a clinically-relevant biologically effective dose (BED) of 101.3 Gy [Overall, this acute increase in osteoclast activity leads to the hypothesis that osteoclast inhibition utilizing a bisphosphonate  could prevent or reduce RT-induced bone loss and prevent fractures . In 2010101.3 Gy . However101.3 Gy . Rats trSeveral of the above-noted rodent studies utilized primarily histologic stains to demonstrate changes in osteoclasts, as well as trabecular number, density, and cortical thickness. Micro-CT also improves upon the evaluation of trabecular volume/number and cortical thickness, but is limited to small regions of interest. A novel three-dimensional (3D) cortical mapping algorithm was developed that could easily be scalable to larger CT scans, multiple bones simultaneously, and provide both quantitative and qualitative information on RT-induced bone changes ,61,62. In the creation of this algorithm, the entire vertebral column of 16 rhesus macaques nonhuman primates (NHPs) were assessed before and after these animals had received whole chest irradiation to 10 Gy to the midplane in a single fraction, delivered with 6-MV X-rays in an Anterior-Posterior / Posterior-Anterior (AP/PA) fashion at a dose rate of 6 Gy/min . All NHPFor each CT scan, the bony anatomy was segmented using automated thresholding techniques and manual editing in Mimics  for Windows. Individual contours of each vertebral body were constructed in treatment planning software and these contours were then utilized to construct 3D surfaces containing over 2000 orthogonal vertices using Stradwin software  for Windows. The global cortical bone density for each CT scan was used and collected density measurements at each vertex. For both skeletally mature and immature animals, the resulting cortical thickness maps showed a significant infield cortical thickness loss at the first imaging time point, 2 months post RT . After tin vivo changes given the close correlation of our findings with histologic photomicrographs. This study demonstrated that in-field RT-induced bone changes occur rapidly, and are unlikely to be repaired. Additionally, it illustrated that our mapping approach was truly reflective of This same cortical thickness mapping approach was applied to a retrospective database of lung cancer patients with peripheral lung tumors receiving SBRT to estimate changes in rib cortical thickness. Patients were treated to 50 Gy at 5 to 10 Gy per fraction. Baseline CT planning scans were compared to 3-month post-SBRT follow-up scans. Using iterative closest point (ICP) registration in MATLAB , one can register received radiation doses to the surface of each rib to the corresponding cortical thickness map [By 3 months post SBRT, we were able to detect rapid cortical thickness loss in field. Furthermore, we were able to describe a continuous dose–response relationship. Regions of ribs that received 10 Gy or more exhibited significant cortical thickness loss at ~7%, with a range of 4% to 11%, but with every additional 10 Gy interval, the degree of cortical thickness loss increased. Regions receiving 40 Gy or more had the greatest cortical thickness loss at 18%, with a range of 15–22% loss. In areas of the ribs receiving >30 Gy, approximately 92% of treated patients developed significant cortical thinning by 3 months, with RIRF occurring as early as 6 months post RT [Utilizing this mapping approach with lower doses per fraction, cortical thinning was once more correlated with total dose received by bone. The CT scans from 23 anal cancer patients receiving 53–58 Gy at 1.8 Gy per fraction were retrospectively analyzed at baseline and <4 months post RT. Again, significant early cortical thinning (< 4 mo) was noted in the intertrochanteric crest, and femoral neck. The femoral neck volume of bone receiving 40 Gy was a significant predictor of focal cortical thinning of 30% or more . ORN of the mandible after head and neck RT is a significant issue affecting cancer patients who receive RT as part of their curative treatment . Over 60The pathogenesis of mandible ORN is thought to be secondary to a combination of tissue hypoxia, hypocellularity, and vascular changes . While tBased on these preclinical data utilizing mouse and NHP models, as well as retrospective clinical studies, a clinical protocol to assess and potentially prevent SBRT-induced bone loss has been developed and activated. For this ongoing trial, the aim is to determine if single dose prophylactic suppression of early RT-induced osteoclast activity with bisphosphonates diminishes or prevents RT-induced cortical bone thinning, fracture, and pain. As demonstrated in The primary objective is to assess the percent change in bone mean cortical thickness within regions receiving 30 Gy or more at 3 months post SBRT. Secondary endpoints will also assess cortical thickness change at 6, 9 and 12 months after SBRT. The percent change in mean cortical thickness will be compared between patients who received risedronate and those who received placebo. Mean cortical thickness will be measured for the ribs and vertebrae within the irradiated field using our cortical thickness and radiation dose mapping approach. The incidence of RIRF within the treatment field will be recorded. CWP at each time point will also be assessed utilizing a modified Radiation Therapy Oncology Group (RTOG) grading scale. As a biomarker correlate, urinary metabolites of bone breakdown will be assessed at each time point. A variety of urinary biomarkers have been linked to increased osteoclastic activity in osteoporotic populations, and the most frequently tested marker is urinary N-terminal telopeptide crosslinks (NTX) . These bBisphosphonates are generally well tolerated, however there is a low risk of osteoradionecrosis (ORN) of the mandible. Amongst patients on chronic risedronate therapy, there was a ~1–12%  risk of There is no reported increased incidence of toxicity in patients receiving chest RT. Concurrent bisphosphonates are not contraindicated prior to chest SBRT. Importantly, none of the patients in this study are receiving head and neck irradiation. Similarly, patients with dental operations involving manipulation of bone within the last 6 months are excluded. n = 2, with peripheral lung tumors who were incidentally taking bisphosphonates when they were treated with lung SBRT. We found no increased risk of any toxicity from concurrent SBRT and bisphosphonate therapy. Furthermore, on cortical thickness mapping, these patients had minimal cortical thinning of the ribs near the irradiated site .For this reason, several alternative strategies for the prevention of RT-induced bone toxicity have also been explored. These include the use of parathyroid hormone (PTH), sclerostin antibody, amifostine, angiogenesis manipulation, anabolic weight loading, and antioxidant dietary supplementation. As noted, anabolic approaches are strong candidates for the prevention of RT-induced fractures due to the known effects on osteoblasts . Based oKang and colleagues also investigated the use of PTH to mitigate the effects of RT on bone using a murine model of distraction osteogenesis . Mice weWhile preliminary and published data on PTH is promising, there is also a concern  that PTH may induce osteosarcomas. This has been shown in limited animal models and case reports in humans with primary hyperparathyroidism leading to prolonged PTH elevation ,78,79. Tin vitro study of rat bone marrow stem cells with or without radiation and amifostine showed that the addition of amisfostine to irradiated cells decreased the amount of reactive oxygen species and DNA damage and increased the levels of cellular proliferation, which is promising [in vivo rat model, by reducing DNA damage, amifostine may decrease the differentiation of osteoclast precursor cells into differentiated osteoclasts. Furthermore, they noted decreased lacunar resorption pits in rodents treated with RT and amifostine than those with RT alone [Amifostine is a radio-protective agent that acts by scavenging free radicals generated by ionizing radiation. A recent romising . AdditioRT alone . These findings show promise that amifostine may be able to reduce RT-induced bone damage. However, there are substantial challenges to this strategy that need to be overcome for routine clinical use for this indication. First, amifostine must be administered intravenously shortly before the delivery of RT, which presents logistical challenges. Furthermore, substantial side effects have been reported in human clinical trials of amifostine, including a Phase III trial of amifostine that was terminated early due to severe toxicity in 41% of patients. A review of the literature in the report of the terminated trial noted an overall 27% rate of amifostine discontinuation due to severe adverse events . Howeverin vitro study, they found that RT induced substantial dysfunction in endothelial progenitor cells, including the downregulation of vascular endothelial growth factor A (VEGF-A), which is a potent angiogenic agent [Angiogenesis is a critical component of bone modeling and remodeling. Tong and colleagues hypothesized that the effects of RT on bone marrow-derived endothelial progenitor cells may also impact the resorption of bone after RT. In an ic agent . Howeveric agent . This maSufficient mechanical loading is critical for proper bone development, leading to increased bone formation and decreased bone loss . HoweverFinally, dried plums are rich in antioxidants, and have been shown to prevent bone loss in murine osteoporosis models. However, until recently, it was not as well studied for RT-induced bone loss. In a murine model, Schreurs et al. showed that dietary supplementation with dried plums decreased the expression of multiple genes involved in bone resorption, including RANKL and TNF-a . AdditioIn summary, bone is a radiosensitive structure that is often unavoidably included in standard RT fields. RT can result in lasting pain, functional impairment, and decreased QOL, even at standard fractionation doses. As high-dose RT for oligometastatic disease becomes more standard in current practice, and as cancer-specific survival continues to improve, we can only expect an increase in the incidence of RT-induced bone toxicity. Preclinical animal models including murine and NHP studies have been crucial to our understanding of how irradiation affects the complex processes governing the bone microenvironment. Furthermore, these animal models have been instrumental to the creation and implementation of new translational clinical trials with therapeutic targets.


Shortness of breath is a common complaint among patients in emergency medicine. While most common causes are usually promptly identified, less frequent aetiologies might be challenging to diagnose, especially in the pre-hospital setting. We report a case of prehospital dyspnoea initially ascribed to pulmonary oedema which turned out to be the result of profound metformin-associated metabolic acidosis. This diagnosis was already made during the prehospital phase by virtue of arterial blood gas measurement. Pre-hospital measurement of arterial blood gases is therefore feasible and can improve diagnostic accuracy in the field, thus avoiding unnecessary delay and potential harm to the patient before initiating the appropriate therapeutic actions. Shortness of breath (SOB) is one of the most common complaints among patients in prehospital emergency medicine . Most co2 65%: pH 6.845, PaCO2 1.95 kPa, PaO2 27.7 kPa, HCO3−—2.5 mmol/L, lactate 17.0 mmol/L).The emergency medical call centre (EMCC) dispatched an advanced life support ambulance to take care of a 71-year-old woman who had called complaining about SOB. Upon arrival, prehospital providers noted shallow breathing with a respiratory rate of over 35 breaths per minute. Lung fields were clear to auscultation and the patient showed no sign of cyanosis. There was no haemodynamic compromise. The Glasgow Coma Scale was 15 with no focal deficit and the patient was normothermic. Given the respiratory distress, paramedics initiated oxygen therapy using a high-flow non-rebreather mask. Suspecting acute heart failure (AHF), they asked the EMCC to dispatch a medical mobile unit called ‘Service Mobile d’Urgence et de Réanimation’ (SMUR). These units are staffed by an advanced paramedic and a physician and can be dispatched to assist an ambulance in case of life-threatening emergencies . These SWhile AHF was suspected at first, the profound metabolic acidosis revealed by the ABG analysis was not compatible with the clinical picture. Indeed, the patient was not in cardiogenic shock, and a diagnosis of lactic acidosis secondary to metformin intoxication was thereafter suspected. Intravenous hydration was started and bicarbonates were administered. Since the patient showed signs of respiratory distress and exhaustion, NIV was initiated.2 according to the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation. This kidney injury was secondary to the pre-renal disease on severe dehydration  and sepsis, with consequent acute tubular necrosis. The patient showed no sign of hepatic impairment nor hypoxaemia. There was no hint that the patient’s illness could have been secondary to suicidal or accidental biguanide ingestion. The diagnosis of profound metformin-associated metabolic acidosis secondary to renal failure was definitively established at this stage. Despite aggressive hydration and bicarbonates infusion, the patient remained oligo-anuric with severe metabolic acidosis and showed signs of respiratory exhaustion finally requiring endotracheal intubation. She was admitted to the intensive care unit (ICU) for haemodynamic and respiratory management. The decision to start haemodialysis was finally made basing on the severity of acidosis and lactate level, coexisting renal failure and failure to improve with bicarbonate therapy [In-hospital investigations confirmed severe lactic acidosis and acute kidney injury KDIGO stage 3 [ therapy . The patSOB is a major complaint among pre-hospital and ED patients, accounting for over 3.9 million visits in 2017 in the United States . Most ofOur case exemplifies how pre-hospital ABG measurement can easily improve diagnostic accuracy in such cases. In our prehospital system and in many European countries, trained physicians can be dispatched by EMCCs to provide medical care in the field , and canAbsolute contraindications to ABG sampling can usually be clinically identified easily , and serious complications  are rare. Minor complications  can be easily managed and anticoagulants or antiplatelet agents do not represent absolute contraindications but often require only increased vascular pressure. Although venous sampling could lead to misdiagnosis, providers can be trained to recognize such samples.In this particular case, onsite ABG analysis allowed the physician to suspect metformin-associated metabolic acidosis and start the appropriate treatment even in the pre-hospital setting. While previous studies have failed to show a general positive effect of prehospital ABG on diagnostic accuracy, it can be of significant benefit in specific situations . MoreoveMetformin-associated metabolic acidosis is a rare but well-known complication of metformin treatment. Its incidence was calculated to be 4.3 cases per 100,000 patient-years . When itAlthough the main cause of the lactic acidosis found on ABG analysis was rapidly identified in this case, other potential aetiologies should be always considered. These include severe sepsis, shock, anemia, seizures, hypoxemia, carbon monoxide or cyanide poisoning, ethylene glycol or salicylates intoxication, alcoholism and liver disease, beta-agonist medication or profound thiamine deficit . In our The role of bicarbonates infusion is still debated in metformin-associated metabolic acidosis as in other causes of anion gap metabolic acidosis . In our This case report shows that prehospital ABG measurement is feasible and can improve diagnostic accuracy in the field. In this particular case, ABG analysis enabled the prehospital physician to make the correct diagnosis in a timely manner, thus allowing the appropriate treatment to be initiated before hospital admission. Unnecessary delay and potential harm to the patient were thereby avoided.


Older adults may exhibit symptoms of Post-Traumatic Stress Disorder (PTSD) after a traumatic event, even if they do not meet the criteria for a full diagnosis. This sub-clinical PTSD affects 7-15% of older adults and is associated with elevated depressive symptoms, suicidal ideation, and poorer physical health. Individuals with sub-clinical PTSD often experience worsening symptoms, resulting in a full diagnosis of PTSD, albeit late onset. This evaluation informs the Veterans Health Administration (VHA)’s understanding of the developmental course of PTSD in older Veterans. VHA administrative data were used to examine health characteristics and service utilization in the five years before and five years after the initial VHA documentation of PTSD. We identified a cohort of Veterans , alive and with at least 1 encounter in all evaluation years, with a first PTSD diagnosis documented between ages 50-59, a cohort outside of the average age of diagnosis, but before Medicare eligibility. We compared periods before and after diagnosis across different ages of first diagnosis . Veterans diagnosed at later ages (55-59) had a greater number of mental and physical health conditions. Increasing VHA use preceded a PTSD diagnosis in both groups, but the increase was steeper among those diagnosed at 55-59. Future analyses will compare these patterns to those of Veterans diagnosed at younger ages. Findings from this work may provide a profile of persons at risk for late-life PTSD for use in targeting interventions earlier to reduce the risk of developing worsening PTSD symptoms in late life.


Effective surveillance and response systems are vital to achievement of disease control and elimination goals. Kenya adopted the revised guidelines of the integrated disease surveillance and response system in 2012. Previous assessments of surveillance system core and support functions in Africa are limited to notifiable diseases with minimal attention given to neglected tropical diseases amenable to preventive chemotherapy (PC-NTDs). The study aimed to assess surveillance system core and support functions relating to PC-NTDs in Kenya.A mixed method cross-sectional survey was adapted involving 192 health facility workers, 50 community-level health workers and 44 sub-national level health personnel. Data was collected using modified World Health Organization generic questionnaires, observation checklists and interview schedules. Descriptive summaries, tests of associations using Pearson’s Chi-square or Fisher’s exact tests and mixed effects regression models were used to analyse quantitative data. Qualitative data derived from interviews with study participants were coded and analysed thematically.Surveillance core and support functions in relation to PC-NTDs were assessed in comparison to an indicator performance target of 80%. Optimal performance reported on specimen handling , reports submission  and data analysis  at the sub-county and county levels respectively. Facilities achieved the threshold on reports submission (84%), reporting deadlines (88%) and feedback (80%). However, low performance reported on case definitions availability (60%), case registers (19%), functional laboratories (52%) and data analysis (58%). Having well-equipped laboratories , PC-NTDs provision in reporting forms  and surveillance training  were associated with higher odds of functional surveillance systems. Challenges facing surveillance activities implementation revealed through qualitative data were in relation to surveillance guidelines and reporting tools, data analysis, feedback, supervisory activities, training and resource provision.There was evidence of low-performing surveillance functions regarding PC-NTDs especially at the peripheral surveillance levels. Case detection, registration and confirmation, reporting, data analysis and feedback performed sub-optimally at the facility and community levels. Additionally, support functions including standards and guidelines, supervision, training and resources were particularly weak at the sub-national level. Improved PC-NTDs surveillance performance sub-nationally requires strengthened capacities.The online version contains supplementary material available at 10.1186/s12889-021-10185-1. Health systems building blocks derived from the World Health Organization (WHO), work in concert with one another and are key to achieving health system strengthening . Health Strengthening national disease surveillance and response systems requires periodic evaluation of the structure, functions and attributes of the system to review strengths, weaknesses, and opportunities for improvement . In KenyNTDs mainly burden health systems at the sub-national level . Hence, The IDSR strategy is comprehensive and comprising of diverse activities to enable effective disease detection to inform appropriate public health action. Four principal elements namely structural, core activities, support activities and attribute functions constitute the IDSR framework. However, this study delineated and focused on the surveillance core and support functions, which form the main driving force to implement the strategy effectively. In addition, bearing in mind the consolidated principle of the IDSR strategy that aims to accommodate all diseases under surveillance, this study assessed the performance of surveillance core and support activities specific to neglected tropical diseases. Assessment of the surveillance functions relating to PC-NTDs was based on components of the system as documented in the WHO guideline . DefinitKenya is situated in East Africa and bordered by Uganda to the West, Somalia to the East, South Sudan and Ethiopia to the North and Tanzania to the South Fig. .Fig. 1MThe country comprises of 47 administrative counties and up to 290 sub-counties. The study areas included endemic counties with at least one sub-county having three co-endemic PC-NTDs of known prevalence Table  24]. Ac. Ac24]. The study targeted healthcare personnel responsible for collection and transmission of surveillance data at the following three levels;Community level: Community health extension workers (CHEWs) responsible for compiling and reporting disease data and assigned to fully functional community health units (CHUs) linked to selected health facilities.Health facility level: Healthcare facility workers (HFWs) in both public and private facilities responsible for collection and transmission of surveillance data. HFWs were drawn from various facility levels including dispensaries (level 2), basic primary health care facilities (level 3), primary care hospitals (level 4) and county referral hospitals (level 5) that reported a high-threshold of PC-NTD cases in the one-year surveillance period.Sub-national health management teams: Healthcare personnel at the sub-county and county levels responsible for collation and transmission of surveillance data reported from the healthcare facilities in the given surveillance period. Also included in this category were key healthcare stakeholders responsible for decision-making and overseeing surveillance and response related activities including county directors of health, county epidemiologists, county NTDs coordinators and county health information managers.At the community level, community health volunteers complete relevant reporting forms either monthly or biannually to capture the monthly services offered, health and demographic information of the households. These reports are submitted to CHEWs to undertake data verification, consolidation and data summaries for onward submission to the health facility chalkboard and further to the upper levels for entry into the DHIS2 . The reqA mixed method cross-sectional survey approach was used to assess the IDSR system surveillance functions comprising; core and support functions. The study involved the concurrent collection of both quantitative and qualitative data. The convergence method employed was aimed at ensuring detailed data was captured from the study participants while giving equal importance in terms of analysis and comparison to both types of data . SurveilStudy areas were purposively sampled from the Rift Valley, Eastern and Coastal parts of Kenya, which are regions endemic of fully mapped PC-NTDs. These regions were selected to enable detailed assessment of NTDs surveillance and response activities within the IDSR system framework. Within the three regions, ten 10) administrative counties prevalent of at least three or more PC-NTDs were purposively sampled. Furthermore, within the counties, nineteen (19) sub-counties reporting a high number of PC-NTD cases in the one-year surveillance period (2017) were selected (Table 0 adminisn = 19) and county disease surveillance and response coordinators (n = 10). Furthermore, fifteen (15) key stakeholders overseeing disease surveillance activities at the sub-national level were also enrolled. Purposive sampling technique was largely preferred since it enabled careful selection of specific “information rich” participants across the surveillance levels to gain extensive knowledge on their experiences ,  and  respectively. Of the facilities with a functional health facility-based surveillance system, 64% (104/161) confirmed to report PC-NTDs through this system and up to 97% of respondents reported that it was important to have a PC-NTD surveillance system at the facility level. Majority  of facility respondents were well aware of PC-NTDs prevalent in the region. Twenty-three percent (45/192) of facility respondents identified Schistosomiasis and Soil Transmitted Helminths as the most common co-endemic conditions while 22% (42/192) reported Lymphatic Filariasis, Schistosomiasis and Soil Transmitted Helminths to commonly co-occur at any given time. At the community level, 88% (44/50) of respondents were aware of the PC-NTDs prevalent in the study regions and 80% of them had identified and reported at least one PC-NTD case at the community level in the previous year.Eighty-four percent (161/192) of facility respondents reported having a functional health facility-based surveillance system with the capacity to detect, confirm, report, analyse and interpret surveillance data to inform response actions. Availability of a laboratory equipped to confirm PC-NTDs cases, provision for PC-NTDs in the reporting forms and training of personnel on disease surveillance were associated with higher odds of having a functional health facility-based surveillance system  and  respectively.Results showed that 84% (162/192) of respondents were aware of deadlines for submission of PC-NTDs surveillance reports at the facility level with 88% of them confirming compliance with the reporting deadlines. Health workers of the nursing cadre (48%) were solely responsible for preparing PC-NTDs surveillance reports at the facility level. Further, 81% of facility respondents confirmed, “zero” reporting was undertaken when there were no PC-NTD cases to report at any given month. Findings from a logistic regression model assessing the predictors of “zero” reporting of PC-NTDs showed that respondents with longer years of work experience had higher odds of undertaking “zero” reporting compared to those who had served fewer years (Table The common channels for surveillance reports submission utilised by respondents at the facility level were through mobile phone short message services (SMSs) (81%) for weekly reporting and in person report submission (73%) for monthly summary reports. At the community level, 82% of respondents reported that the channel mostly used to transmit PC-NTDs monthly surveillance reports was in person submission of hardcopy forms to the health facilities. Respondents at the community level also reported using phone calls (64%) and mobile phone SMS (48%) to transmit surveillance data during weekly reporting. Respondents across the surveillance levels reported challenges using the available reporting channels;“Submitting reports via the DHIS2 portal is at times challenging given the inconsistent internet connectivity in the area … most times we have to incur the expenses resulting from purchase of internet bundles so as to access the portal during report submission” – KII#003 (West Pokot County).“Long distances between the health facility and the next reporting level … and poor terrain in the region pose a challenge to delivering hardcopy monthly summary reports within the required time” – SCDSRC#001 (Baringo County).Health personnel mainly recommended improving PC-NTDs reporting within the IDSR system through provision of adequate resources to facilitate surveillance data reporting. Adopting electronic reporting tools through use of computers and mobile phone devices, and provision of financial incentives to cover for airtime, internet and transport costs;“If we had an electronic reporting system right from the facility level it would ease reporting and eliminate the burden of having to physically submit monthly summary reports” – KII#001 (Baringo County).Respondents further recommended provision of adequate reporting forms at any given time to ensure timely compilation of surveillance reports. In addition, providing improved monthly summary reporting forms with the inclusion of PC-NTDs to enhance their priority in surveillance reports;“The current reporting forms hardly include most of the NTDs … this makes it difficult to report the cases especially in the monthly summary reports … it would be better if most of the NTDs common in the region would be included in the reporting forms” – HFW#112 (Kitui County).Further recommendations alluded to enhanced training amongst health facility workers on the use of reporting tools and the need for health workers sensitisation on the benefits of effective surveillance reporting;“We need periodical training and capacity building on NTDs surveillance activities...health workers sensitisation on NTDs will improve reporting of the cases through the surveillance system” – HFW#089 (Kilifi County).p = 0.002],  and  respectively  respectively. On the contrary, supervision of surveillance activities was associated with 70% reduction in the odds of conducting data analysis at the facility level .Surveillance data analysis for PC-NTDs at the health facility level was mainly either based on age and locality of the individual (27%), age and gender of the individual (23%) or solely based on the individuals’ age (24%). Among health facilities that conducted data analysis, 44% 49/111) performed trend analysis based on PC-NTDs surveillance data collected in the previous year. Availability of PC-NTDs case definitions, presence of disease-specific case registers and receipt of feedback on surveillance reports were associated with higher odds of conducting analysis of surveillance data at the facility;  and  respectively  of respondents reported to receive feedback relating to PC-NTDs from the facility level and 32% indicated that it took more than a week to receive the feedback reports. At the facility level, 37% (70/192) of facilities received feedback on PC-NTDs reports submitted to the next surveillance level in the previous one-year surveillance period. Of this fraction, 39% of the facilities received 1–2 feedback reports while 41% received at least three reports from the higher levels. The ability to meet reporting deadlines and to conduct data analysis at the facility level were associated with increased odds of receiving feedback on surveillance reports; ,  and  respectively. Respondents reported that having in place alternative PC-NTDs surveillance strategies within the existing IDSR system would influence their satisfaction with the system;“We require a behavioral surveillance system to better understand community dynamics for effective active case finding of neglected diseases … these are diseases (NTDs) associated with a lot of stigma, therefore, making it difficult to capture certain cases with a majority of those suffering from example elephantiasis shying away from the public eye” – KII#009 .Logistic regression analysis assessing the predictors of satisfaction with PC-NTDs surveillance and response activities indicated that the facility level, adequacy of forms for reporting PC-NTDs and feedback on submitted PC-NTDs surveillance reports were associated with increased odds of satisfaction with PC-NTDs surveillance and response activities in the endemic region; [AOR = 3.04, 95% CI: 1.77, 5.23; There were eleven a priori identified main themes with up to 62 emerging sub-themes, which were derived based on recommendations to improve PC-NTDs surveillance and response within the existing IDSR system according to health workers’ perspectives Tables  and 13. The study assessed surveillance core and support function performance based on specific measurable indicators. These indicators were compared to the gold standard IDSR indicator performance target of 80% across all surveillance levels –46.The idea of an integrated disease surveillance and response framework was propositioned by WHO based on the sub-optimal performance of vertical surveillance systems characterised by unstandardised case definitions, numerous reporting forms, limited laboratory capacity and inadequately trained human resource . OptimalIn the current study, an assessment of IDSR surveillance functions considering PC-NTDs revealed sub-optimal system performance, which hampered adequate detection and prompt implementation of appropriate public health actions. Focused efforts to improve specific surveillance functions in connection with PC-NTDs are paramount for effective identification of disease foci to inform targeted treatment and halt disease transmission. However, a review of existing literature identified unwavering focus on notifiable diseases in assessing the performance of surveillance systems , 53–57. Efficient case detection is dependent on the availability of case definition guidelines and well-trained health personnel on utilising the available case definitions . AvailabHealth providers reported lack of specific case registers for PC-NTDs with the exception of facilities designated as treatment sites. Similarly elsewhere, health facilities lacked dedicated registers to capture specific diseases during investigation , 60. DisSome PC-NTDs but not all are reported through the health management information system (HMIS), for instance, data on Soil transmitted helminths, Guinea worm disease and Schistosomiasis are captured at the peripheral level. However, data on Lymphatic Filariasis, Leishmaniasis, Onchocerciasis, Trachoma among other PC-NTDs are not incorporated in the health facility physical registers and the HMIS . These dOverall, current study findings mainly identified use of paper-based reporting systems especially at the health facility level. Similarly elsewhere, weekly surveillance reports prepared at the health facility level were mostly submitted in paper format , 60, 62.Findings revealed that surveillance data analysis at the peripheral levels of PC-NTD endemic regions was inadequate. Comparably, routine data analysis was conducted in only a third of the health facilities surveyed from selected WHO African Region countries . RoutineOur study findings identified limited feedback to peripheral levels on PC-NTDs surveillance data submitted to the higher surveillance levels. Several other studies have reported low feedback from national or regional levels to the lower surveillance levels , 46, 59.Health personnel reported limited outbreak preparedness at the sub-national level regarding adequacy of supplies to respond to probable PC-NTD epidemics in Kenya. Another study showed regional surveillance levels lacked adequate outbreak preparedness supplies due to insufficient budgetary allocation for epidemic emergencies . Poor buInaccurate case registration at health facility levels was due to inconsistent use of the standard case definitions similar to our study findings on irregular use of PC-NTDs case definitions . SimilarSupervision of surveillance activities at the peripheral levels of PC-NTD endemic regions in the current study was limited, similar to what has been reported in other studies , 59, 71.Similar to present study findings, most surveillance staff elsewhere were trained on disease surveillance activities . HoweverInsubstantial funding in support of surveillance activities has been identified as a major hindrance to optimal functioning of surveillance and response systems , 59. MorHealth providers outlined various logistical challenges limiting surveillance performance in PC-NTDs endemic regions in Kenya. Comparably, elsewhere, weak technical and logistical capacities hindered IDSR system implementation . ConsequThe main strength of the current study was the use of a mixed methods survey approach, which gave an in depth understanding of the issues relating to PC-NTDs surveillance within the existing IDSR system based on users’ perceptions. However, there were several limitations to this study, firstly. In the first instance, study participants were required to recount specific surveillance activities conducted in the past, a situation that could have introduced instances of recall bias. This was minimised by limiting the assessment to a one-year surveillance period to ensure accurate recollection of specific surveillance activities. Secondly, the study was based on individual perceptions, which may have influenced some of their responses to what they considered socially desirable. To overcome this limitation, the study participants’ responses were supplemented by direct observations. Thirdly, the study focused on assessing health personnel perspectives at the sub-national, which provides the first contact of health services to NTDs afflicted communities. However, stakeholder perspectives at the national level are crucial in the policymaking process and further investigations need to be undertaken in subsequent studies. Fourth, logistical challenges relating to inaccessible study sites, which were characterised by very poor terrain, harsh climatic conditions and personal security concerns, were mitigated through use of telephone interviews. Lastly, there were limited number of studies assessing PC-NTDs surveillance activities to make sufficient comparisons with the current study findings. However, this study provides a comprehensive overview of critical recommendations to address key challenges facing PC-NTDs surveillance within the IDSR system.The sub-national level is the crux of IDSR implementation with the capacity to provide adequate response to public health conditions. Over reliance on donor-initiated PC-NTDs control programmes in endemic regions leads to reluctant control efforts by the sub-national health systems. Principally, strengthened health information systems are a product of quality data generated through effective and well-functioning disease surveillance and response systems. Surveillance core and support functions regarding common disease conditions seem to perform optimally and have been documented extensively in Kenya , 32. HowThe sub-national levels are compelled to strengthen PC-NTDs case-based surveillance to ensure adequate case confirmation of suspected cases and early detection of probable outbreaks as a critical measure to control and eliminate the diseases. The study further recommends periodic and objective assessment of the surveillance system focusing on PC-NTDs. The sub-national level of the healthcare system provides an adequate platform for operational activities of disease surveillance and response systems as opposed to the national levels that provide a much more strategic platform for decision-making . TherefoAdditional file 1.


In cases of multiple lung cancers, individual tumors may represent either a primary lung cancer or both primary and metastatic lung cancers. In this study, we investigated the differences between clinical/histopathological and genomic diagnoses to determine whether they are primary or metastatic. 37 patients with multiple lung cancers were enrolled in this study. Tumor cells were selected from tissue samples using laser capture microdissection. DNA was extracted from those cells and subjected to targeted deep sequencing. In multicentric primary lung cancers, the driver mutation profile was mutually exclusive among the individual tumors, while it was consistent between metastasized tumors and the primary lesion. In 11 patients (29.7%), discrepancies were observed between genomic and clinical/histopathological diagnoses. For the lymph node metastatic lesions, the mutation profile was consistent with only one of the two primary lesions. In three of five cases with lymph node metastases, the lymph node metastatic route detected by genomic diagnosis differed from the clinical and/or pathological diagnoses. In conclusion, in patients with multiple primary lung cancers, cancer-specific mutations can serve as clonal markers, affording a more accurate understanding of the pathology of multiple lung cancers and their lymphatic metastases and thus improving both the treatment selection and outcome. In patients with synchronous or metachronous multiple cancers, individual tumors may appear as either a primary lung cancer or both primary and metastatic lung cancers. The selection of treatment in such cases is dependent on the resulting characteristics. In patients with multiple lung cancers, the nature of a tumor  can usually be judged on the basis of diagnostic imaging findings, clinical course, and/or pathology. If individual tumors composing multiple lung cancers are histologically inconsistent in terms of histological morphology and/or cellular atypism, the multiple onset of primary cancers is highly likely. However, there are no specific radiological, clinical or histological features that can be utilized to unambiguously distinguish intrapulmonary metastases from multiple primary cancers and the cut diagnosis can be perplexing in the clinical setting. The differing biological activities of tumors allow for prognostic distinctions to be drawn and patients with intrapulmonary metastasis are supposed to have a poorer prognosis. Therefore, it is critically important to develop improved methods for the identification of tumors by exploring new, practical techniques and markers. We have previously demonstrated that as a more precise and clinically applicable method, a comparison of the driver mutation profiles enables elucidation of the clonal origin of tumors and thus facilitates an accurate discrimination between primary and metastatic tumors . HoweverThe study enrolled 37 patients who had undergone surgery for multiple lung cancers in our department between January 2015 and July 2019. Written informed consent for genetic research was obtained from all patients, which was performed in accordance with protocols approved by the institutional review board in our hospital. Histological typing was performed according to the World Health Organisation (WHO) classification (3rd edition) [A serial section from formalin-fixed, paraffin-embedded (FFPE) tissue was stained with hematoxylin-eosin and subsequently microdissected using an ArcturusXT laser capture microdissection system . DNA was extracted using the QIAamp DNA FFPE Tissue Kit . FFPE DNA quality was verified using primers for the ribonuclease P locus. Peripheral blood was drawn from each patient immediately before surgery. A buffy coat was isolated by centrifugation and DNA was extracted from these cells using the QIAamp DNA Blood Mini Kit (Qiagen).A panel covering the exons of 53 lung cancer-related genes see  was desiThe sequence data were processed on standard Ion Torrent Suite Software. Raw signal data were measured using the Torrent Suite version 4.0. The pipeline consisted of signaling processing, base calling, quality score assignment, read alignment to the human genome 19 reference (hg19), mapping quality control and coverage analysis. After the data analysis, the annotation of single-nucleotide variants and indels (insertions and deletions) was performed on the Ion Reporter Server System (Thermo Fisher Scientific). Blood cell DNA extracted from the peripheral blood was used as a normal control to detect variants . Sequencing data were visually analyzed using an Integrative Genomics Viewer.The 37 patients recruited in this study  were divided into different groups according to the following characteristics : 31 maleTwenty nine patients were diagnosed with double or triple primary lung cancers on the basis of histopathological characteristics, including 15 patients with adenocarcinoma–adenocarcinoma, 3 patients with squamous cell carcinoma–squamous cell carcinoma, 5 patients with adenocarcinoma–squamous cell carcinoma and 6 patients with other combinations. In terms of tumor development, tumors developed synchronously and metachronously in 26 and 11 patients, respectively. In patients with metachronous tumors, the tumors were designated as tumor 1 (T1), T2 and T3 in chronological order from the earliest to the latest. In those with synchronous tumors, this designation was based on the order of size from the largest to the smallest.Targeted sequencing was performed on 76 surgically resected tumors and 8 lymph nodes obtained from 37 patients, with their blood cell samples utilized as normal controls. The mean coverage depth was 1411-fold for cancer samples  and 1387-fold for blood cell samples . Sequence analyses detected 314 somatic mutations with an allele fraction ≥1% from 84 cancer lesions (1–54 mutations per tumor) . Among tIn 29 patients, the gene, amino-acid substitution and nucleotide changes that were caused by these somatic mutations within individual tumors composing the multiple lung cancers lacked consistency . Thus, tA 74-year-old man had two tumors in the right upper lobe that were resected through right upper lobectomy. Both tumors morphologically had an irregular surface; thus, they were diagnosed as primary lung cancers A,B. PathA 59-year-old woman presented with 0.7-cm nodules in the right lower lobe 1.5 years after undergoing right upper lobectomy for cancer. The tumors were round and had a smooth surface. Because of their morphology, they were suspected of being metastatic lesions. After 4 months of follow-up, there was no increase in the number of lung lesions, suggesting solitary intrapulmonary metastasis. Subsequently, wedge resection was performed. Although both tumors were pathologically papillary-predominant adenocarcinoma C,D, a leA 74-year-old man presented with tumors measuring 4.0 cm and 1.8 cm in the left upper lobe, so left upper lobectomy was performed A,B. As bLymph node metastasis was detected in five patients with double primary lung cancers . It occuA 74-year-old man, described as case C in the previous section, presented with paratracheal and mediastinal lymph node metastases 1 year after left upper lobectomy F. AlthouA 77-year-old man with lung cancer underwent left lower lobectomy A. One yeA 72-year-old man presented with two tumors in the right lower lobe. Imaging findings suggested double primary lung cancers and right lower lobectomy was performed G,H. PostIn cases of multiple lung cancers, clinical differentiation between primary and metastatic tumors can be difficult, rendering treatment selection challenging. Furthermore, in patients with multiple lung cancers metastasized to the lymph nodes or distal sites, the focus of treatment varies depending on the cancer that has metastasized. Thus, determining the origin of the metastasizing cancer is clinically important. Therefore, we performed lung cancer mutation analysis through targeted deep sequencing and demonstrated that mutations of individual lung cancers are able to provide clonal markers, enabling discrimination of the clonal origin of multiple lung cancers and their metastases.The consistency of mutations across multiple sites, with complete concordance in the position and patterns of base-pair substitutions or indels, cannot be a coincidental phenomenon. Although discordance between two tumors was noted in mutations with an allele fraction <20%, this can be interpreted as tumor heterogeneity . In geneIt is relatively straightforward to diagnose multicentric primary lung cancers of different histological type. However, it is often difficult to differentiate between multiple primary lung cancers and intrapulmonary metastases having the same histological type. In particular, in cases of multiple tumors classified as squamous cell carcinoma (such as cases C–F), differentiation based on pathological features alone is extremely difficult. Even when the morphological and immunohistological features are non-homogeneous among different parts of the tumors , the driver mutation is ubiquitously retained within the tumors of the same clone ,17. TherDetterbeck et al. reviewed the clinical and pathological criteria to distinguish second primary tumors from metastatic tumors . They reWhen selecting treatment methods for multiple lung cancers, it is necessary to consider the cancer type will markedly affect the prognosis. In cases D–F that have lymph node metastasis, a factor responsible for progression to an advanced stage was identified in the two tumors. Furthermore, the tumors exhibit different mutation profiles and PD-L1 staining properties. Therefore, the lesions targeted for treatment and the options selected for subsequent treatment  vary depending on the type of tumor that has metastasized to the lymph nodes. This suggests that accurate understanding of the pathology gained by performing a genetic diagnosis can exert a powerful effect on the clinical outcome. Although the use of immunotherapy has revolutionized the treatment of non-small-cell lung cancer, patterns of immunostaining with PD-L1, a biomarker for treatment response, may vary in tumor cells across individual primary tumors  cases E and F) . At presIn cases of multiple lung cancers, identifying the differences in the mutation profiles of multiple tumors will help determine their clonal origin and enable a distinction to be drawn between primary and metastatic tumors with great specificity, even in cases in which pathological distinction is impossible or equivocal. In addition, performing genetic diagnosis in addition to pathological diagnosis can help obtain a more accurate understanding of the pathology of multiple lung cancers and the lymphatic metastases. This approach may lead to the provision of treatment specifically tailored to the features of individual cases.


Despite the strong evidence associating autoimmune diseasewith lung cancer in particular, it is unclear how the presence of autoimmune diseaseaffects morbidity and mortality in these patients. While some studies suggest that thepresence of autoimmune disease adversely affects cancer-related outcomes,5 other studies have not substantiatedthese observations.6 In the currentissue of JAMA Network Open, Jacob and colleagues7 compared lung cancer survival in patients withand without autoimmune disease and found no difference in survival between these 2groups. Using a retrospective design of patients observed at a single academic centerfrom 2003 to 2019, the authors identified 177 patients with biopsy-proven lung cancerand concomitant autoimmune disease and 219 patients with biopsy-proven lung cancerwithout autoimmune disease. Most patients were followed up for as long as 5 years aftertheir cancer diagnosis. Only 14 and 19 patients in the autoimmune disease and controlcohorts, respectively, were lost to follow-up during this time.A number of studies have demonstrated that patients with autoimmune and chronicinflammatory diseases are at heightened risk of the development of cancer, possibly dueto factors such as aberrant immune function and surveillance,While the baseline demographic features, histopathology, and lung cancer stage ofthe 2 cohorts were similar , the cancer treatment regimens varied.Fewer patients in the autoimmune disease cohort received standard-of-care initialtreatment (126 [69.5%] vs 213 [97.3%]) as well as immunotherapy (8 [4.5%] vs 74 [33.8%])compared with the control cohort. Among the autoimmune disease cohort, the most commonreason for not receiving standard-of-care cancer treatment was poor performance statusand/or frailty. Despite the observed treatment regimen disparities, there was nodifference in progression-free survival by stage between the 2 cohorts. Moreover, in amultivariable Cox proportional hazards model analysis, the only independent factorassociated with worse progression-free survival was increased age, while sex, race,smoking status, and the presence of an autoimmune disease were not significantlyassociated. Similar results were found for the analysis of overall survival andrecurrence in locoregional disease. Taken together, the results of this study suggestthat the presence of an autoimmune disease does not adversely affect cancer-relatedoutcomes, at least during a 5-year follow up period.In a subgroup analysis, the authors also found no difference in survival amongpatients with specific autoimmune diseases  or based on the presence of specific comorbidities. However, these analyses may have beenunderpowered to detect significant differences.A notable strength of the present study design included the comprehensivecharacterization of the 2 cohorts through an extensive medical record review. Althoughthe use of large databases in oncology outcomes research affords the study of largersample sizes and more diverse patient populations, these databases often possessvariations in coding, unrecorded variables, and selection bias as well as incompleteassessment of clinically relevant end points. Thus, although the sample size of thepresent study was relatively small, the small number of patients lost to follow-upprovides reassuring evidence that the present findings are unlikely to be due to chancealone.However, it is unclear whether the results are generalizable to other patientpopulations, namely patients who receive care outside of an academic medical center. Theauthors astutely noted that survival in both groups was better than expected relative tothe general population. It is also conceivable that both lead time bias and length biascontributed to improved survival in patients with autoimmune disease. For instance,patients at risk of autoimmune-associated ILD often undergo screening for ILD withhigh-resolution computed tomography imaging. Length time bias is a particular concern inthis scenario because certain detected cancers may be slowly growing and may haveotherwise never come to clinical attention in the patient’s lifetime hadscreening not occurred.It is also unclear how the differences in treatment strategies between the 2cohorts may have contributed to the observed outcomes. Substantially more patients inthe control cohort received immunotherapy for their cancer, and the reasons for thisdifference are not entirely clear. While the preexistence of autoimmune conditions maypredispose patients to more severe complications from such therapy, this is atheoretical concern and warrants further investigation.In summary, the present study provides compelling evidence that the existence ofan autoimmune condition does not adversely affect survival in patients with lung cancer.Future studies are needed to evaluate whether specific immunomodulatory agents used totreat autoimmune conditions as well as the duration of immunomodulatory therapy affectlung cancer survival rates in these patients. The answers to these questions may helpguide future therapeutic efforts in this area.


Plant Ecological Unit’s (PEUs) are the abstraction of vegetation communities that occur on a site which similarly respond to management actions and natural disturbances. Identification and monitoring of PEUs in a heterogeneous landscape is the most difficult task in medium resolution satellite images datasets. The main objective of this study is to compare pixel-based classification versus object-based classification for accurately classifying PEUs with four selected different algorithms across heterogeneous rangelands in Central Zagros, Iran. We used images of Landsat-8 OLI that were pan-sharpened to 15 m to classify four PEU classes based on a random dataset collected in the field (40%). In the first stage, we applied the following classification algorithms to distinguish PEUs: Minimum Distance (MD), Maximum Likelihood Classification (MLC), Neural Network-Multi Layer Perceptron (NN-MLP) and Classification Tree Analysis (CTA) for pixel based method and object based method. Then, by using the most accurate classification approach, in the second stage auxiliary data ) was incorporated to improve the accuracy of the PEUs classification process. At the end, test data (60%) were used for accuracy assessment of the resulting maps. Object-based maps clearly outperformed pixel-based maps, especially with CTA, NN-MLP and MD algorithms with overall accuracies of 86%, 72% and 59%, respectively. The MLC algorithm did not reveal any significant difference between the object-based and pixel-based analyses. Finally, complementing PCA auxiliary bands to the CTA algorithms offered the most successful PEUs classification strategy, with the highest overall accuracy (89%). The results clearly underpin the importance of object-based classification with the CTA classifier together with PCA auxiliary data to optimize identification of PEU classes. Over four decades of satellite remote sensing application to assessment and vegetation mapping using satellite image processing for quantitative detection of sparse vegetation, distinction of PEUs remains problematic and challenging . GeneralAltogether, this study aims to compare pixel-based and object-based approaches with selected four different classifier algorithms for PEUs mapping. Two specific objectives were addressed: (1) the first stage aimed to evaluate the use of Minimum Distance (MD), Maximum Likelihood Classification (MLC), Neural Network-Multi Layer Perceptron (NN-MLP) and Classification Tree Analysis (CTA) algorithms for pixel- and objectbased methods Fj to identify PEUs. (2) In the second stage, auxiliary data (PCA) were used to make the most accurate PEUs classification map based on the best performing algorithm. This study will eventually provide insights into the classification strategies for the PEUs mapping in the semi-arid rangelands.22.1Semi-steppe rangelands Marjan is located within the Central Zagros in Southwest, Iran. The area covers 7736.24 ha extending from 51°18′53″ to 51°19′12″E and 32°03′56″ to 32°04′05″N and with a mean elevation of 2697.48 m a.s.l . The cli2.2Astragalus verus Olivier (As ve)), (2) PEU2 (Bromus tomentellus Boiss (Br to)), (3) PEU3 ), and (4) PEU4 ). Canopy cover data could potentially be used to identify PEUs from structural, compositional or combination of both, the so-called physionomic-floristic classification, to have a sound and accurate perspective on PEUs. We sampled the four identified PEUs using three replicates, in each of which canopy cover was sampled along three transects of 100 m that were evenly distributed throughout the Marjan. The sampling was systematic-randomly . We collected species-based canopy cover within each quadrat. In each PEU, canopy cover percentage was calculated and the PEU types were named according to their dominant floristic composition. For this purpose, first, the dominant plant species of each PEU was identified and then its accompanying species was determined with having 50% or more canopy cover of previously dominant species cover. Thus, each PEU was named based on a physiognomic–floristic method.Four PEU classes were identified in the study area , namely:2.3https://earthexplorer.usgs.gov/). This date almost represents a peak in the phenological development for the majority of PEUs in the area. Bands uninformative for vegetation mapping  were excluded )4.2For the MD, NN-MLP and CTA algorithms, the object-based strategy outperformed the pixel-based one, while the MLC algorithm revealed no significant differences between both strategies. Hence, these results suggest that with MLC both pixel/object-based methods can be used to classify PEUs with respect to overall classification accuracy. Similarly, in the study of Xie  it was c4.3The second model is based on auxiliary data along with raw bands to obtain the most accurate map of PEUs. PCAs is a linear transformation method, which is valuable in improving PEUs classification accuracy. The results of the second stage  showed cThe purpose of PCA is to reduce the number of dimensions by gathering the most useful variations. When considering the first three PCAs, more than 99% of spectral information is presented, thereby reducing noise and therefore improving classification accuracies . LikewisHere, the similar spectral behavior of PEUs species in the heterogeneous rangelands complicated the accurate identification of the PEUs boundaries. Subclasses of a land cover, such as PEUs as a subclass of rangeland cover, are more spectrally similar than that of a higher hierarchical land cover, especially when medium spatial resolution imagery like Landsat OLI-8 data is used for mapping. Furthermore, since this study was conducted in a relatively arid region, surface reflectance values of PEUs are often mixed with information of background soil reflectance. Bare soil reflectance imposes a significant impact on the spectral behavior of PEUs characterized by sparse vegetation cover, and usually hides the spectral responses of patches of vegetation covers. Likewise, in a related study about land use/cover classification in arid rangelands, Genbatu Ge  observed5Our analysis provides insights into the way advanced classification algorithms can identify heterogeneous vegetation communities with similar spectral behavior and a complex landscape structure. The presented results underline that in a heterogeneous landscape accurate PEUs mapping is determined according to the type of supervised classification, pixel-based or object-based methods, and the choice of the classifier algorithm. Four classification algorithms applied to PEUs classification revealed that the results of the classification algorithms varied considerably, so that the CTA algorithm yielded the most accurate results, followed by MLC and NN-MLP. In addition, the object-based methods clearly outperformed the pixel-based ones. Not all spectral bands offered valuable information in the classifications. Merging auxiliary PCA bands derived from the original Landsat imagery together with the raw bands offered the most valuable information to distinguish PEUs, with an overall accuracy of 89%. The development of a comprehensive classification procedure and deep learning algorithms may stimulate new research directions for accurate PEUs mapping.


Mitochondria are best known for their role in energy production, and they are the only mammalian organelles that contain their own genomes. The mitochondrial genome mutation rate is reported to be 10–17 times higher compared to nuclear genomes as a result of oxidative damage caused by reactive oxygen species during oxidative phosphorylation. Pathogenic mitochondrial DNA mutations result in mitochondrial DNA disorders, which are among the most common inherited human diseases. Interventions of mitochondrial DNA disorders involve either the transfer of viable isolated mitochondria to recipient cells or genetically modifying the mitochondrial genome to improve therapeutic outcome. This review outlines the common mitochondrial DNA disorders and the key advances in the past decade necessary to improve the current knowledge on mitochondrial disease intervention. Although it is now 31 years since the first description of patients with pathogenic mitochondrial DNA was reported, the treatment for mitochondrial disease is often inadequate and mostly palliative. Advancements in diagnostic technology improved the molecular diagnosis of previously unresolved cases, and they provide new insight into the pathogenesis and genetic changes in mitochondrial DNA diseases. Historically mitochondria evolved from a bacterial ancestor of α-proteobacteria and became endosymbionts living inside eukaryotes over one billion years ago . Under nMitochondria are dynamic, constantly fusing and dividing . A majorMitochondria are unique compared to other organelles in animals as they have their own DNA. The mtDNAs are arranged in protein–DNA complexes that are also known as mitochondrial nucleoids which are found in the mitochondrial matrix . Human mMtDNA is packaged into large nucleoprotein complexes named nucleoids, and the major structural component of the nucleoid is transcription factor A, mitochondrial (TFAM). TFAM binds to DNA without sequence specificity and is essential in mitochondrial transcription machinery . The nonMtDNA diseases result in severe multisystem complications during the neonatal phase, adolescence, or, in some cases, during adult onset  with a pMtDNA disorders were initially considered to be uncommon due to the widespread symptoms which involve cardiovascular, neurological, and age-related degenerative diseases. Patients with mitochondrial disease display a cluster of clinical features that can be categorized into a discrete clinical syndrome. Common mitochondrial diseases include Leigh syndrome, Leber hereditary optic neuropathy (LHON), myoclonic epilepsy with ragged red fibers (MERRF), and mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) .The first population-based study of a single pathogenic mtDNA mutation was reported in Finland with the prevalence of MELAS as high as 16.3 in 100,000 . In EnglMT-TL1) gene [MT-TV) gene [MT-CO3) gene [MT-ND5) mutations  [MELAS is one of the most frequently maternally inherited mitochondrial disorders, and it impairs mitochondrial translation and protein synthesis, which results in the inability of mitochondria to meet the energy demand of various organs, eventually causing multi-organ dysfunction . MELAS iL1) gene ,29. OtheL1) gene  and m.16TV) gene , which iO3) gene , and sev3514A>G) ,34,35,363514A>G) ,37.MT-TK) gene  are the most common cause of MERRF, occurring in more than 80% of all cases [MT-TL/tRNA (Leu) (m.3291T>C) [MT-TF/tRNA (Phe) [MT-TP/tRNA (Pro) genes [MERRF syndrome is a rare syndromic mitochondrial disorder (MID) with broad phenotypic but narrow genotypic heterogeneity. Commonly, it is known as a chronic neurodegenerative disease that can manifest in both children and adults. Symptoms of MERRF include myoclonus, myopathy, and spasticity. Mutations in the mitochondrially encoded tRNA lysine (ll cases . Mutatio3291T>C) ,40, tRNA3291T>C) , MT-TF/tNA (Phe) , and MT-o) genes .ND1) (G3460A), ND4 (G11778A), or ND6 (T14484C) genes, which result in a dysregulated complex I of OXPHOS [LHON patients typically present with painless loss of central vision in one eye, followed by loss of vision in the second eye within weeks or months . It predND1) G30A, ND4 , and chronic progressive external ophthalmoplegia (CPEO) . PearsonMethods for detecting mutations in mtDNA do not differ much from those used to determine the primary sequence of any DNA. The first generation of DNA sequencing involved fragmentation and detection of radiolabeled DNA, and these protocols quickly improved over the years with parallel sequencing techniques . In the Recently, rolling circle amplification (RCA) was used to amplify mtDNA in a heritability analysis study of 35 individuals . RCA, a This review outlines the current advances in in vitro mitochondria manipulation, which allows researchers to understand the pathogenic implication and therapeutic potential of mtDNA mutation. The strategies involve either removing the detrimental mtDNA by transferring healthy mitochondria or targeting specific mtDNA sequences that cause mitochondrial disease .Mitochondria transfer technologies focus on introducing exogenous mitochondria to a recipient cell. In this technique, the mtDNA is not manipulated. MtDNA replacement technology is a more specific and targeted method that can generate non-native mtDNA sequences or repair the sequences of existing mtDNA to shift the mtDNA heteroplasmy ratio.The mitochondrion is an endosymbiotic organism with partial nuclear independence, allowing it to be exchanged between cells . The traExogenous mtDNA molecules are either incorporated into mammalian cells or into empty mitochondria and then are introduced into mammalian cells via endocytosis (Patent No: EP3067416A1). Viable isolated mitochondria can be internalized by simple co-incubation, which involves macropinocytosis. However, the internalization is only for a short duration of time . AnotherThe mitochondrial inner membrane that selectively transports molecules into the mitochondrial matrix is impermeable to hydrophilic molecules. Exogenous genes with functional protein expression are transported into mitochondria to compensate for mitochondrial dysfunction as a consequence of mtDNA mutations.Labeled DNA oligonucleotides are transferred into the mitochondrial matrix using peptide nucleic acid (PNA) . PNA hasSaccharomyces cerevisiae [Chlamydomonas reinhardtii [Biolistic transformation utilizes a helium shockwave to deliver DNA on microscopic metal particles that is incorporated into mtDNA via active homologous recombination . To daterevisiae  and Chlanhardtii  are the Gene editing in the mitochondria is based on the concept of using the inefficient double-strand break repair system in mitochondria and introducing endonucleases to degrade the mutant mtDNA and to repopulate with wild-type mtDNA. Pathogenic mtDNA mutations are generally heteroplasmic with the presence of pathology characteristics when the ratio of mutated mtDNA exceeds a certain threshold.Recently, Gammage et al. improved their previous findings on zinc finger nucleases (ZFN) to target and cleave predetermined loci in mtDNA . They geMitochondrially targeted transcription activator-like effector nucleases (mitoTALENs) are used to cleave specific sequences in mtDNA with the goal of eliminating mitochondria carrying pathogenic point mutations. This approach was recently explored in two clinically important mtDNA point mutations associated with mitochondrial disease, which include myoclonus epilepsy with ragged red fibers (MERRF) and MELAS/Leigh syndrome . The mutAla gene mutations was the first mouse model with a heteroplasmic pathogenic mtDNA mutation generated. This mouse is associated with tRNAAla instability and a mild cardiac phenotype at old age [Ala characterized by cytochrome C oxidase (COX)-deficient fibers that are associated with myopathy and impairment of OXPHOS [MitoTALEN specific for the mtDNA region harboring the m.5024C>T mutation was cloned into an adeno-associated virus and phage (AAVP) vector to test its role in regulating mtDNA heteroplasmy in vivo . A mouse old age . It resef OXPHOS . A signif OXPHOS .Research and trial of mitochondrial manipulation raise several bioethical issues. The clinical use of mitochondrial modification involves germline modification. Unlike somatic modification, these manipulated genes are transmitted to the offspring. The ability to freely manipulate genes hinders the future generation from receiving an unmanipulated gene pool. Furthermore, children born from a mitochondrial replacement procedure would have a genetic link to three people: their parents and the donor ,73.MT-ND6 gene, associated with Leigh syndrome [Predictive tests such as pre-implantation genetic diagnosis (PGD) and pre-natal diagnosis (PND) are available to avoid mtDNA disorder transmission. The two reproductive options that are well documented for mitochondrial donation in women with pathogenic mtDNA mutations who wish to reduce the risk of mitochondrial disorder in their children are maternal spindle transfer (MST) in unfertilized oocytes and pronuclear transfer (PNT) in zygotes . The firsyndrome .The debate about the ethics of germline modification is inevitable. The manipulation techniques which involve egg donation remain controversial and pose a potential risk to the donor. Since the transmission of mtDNA disorders is complex and hard to predict, a safe and effective technique with appropriate information and support is pertinent to the affected patient. Significant precaution measures should be taken in techniques involving altering genes; however, the goal of treatment is to alleviate suffering of the patient, and their families should also be considered.MtDNA disorders are among the most common inherited human diseases, and their prevalence was shown to be influenced by demographic and genetic factors . The hetSeveral mtDNA diseases are due to either dysfunction in mitochondrial replication or translation, which results in mtDNA mutation(s) or a mutation in nuclear-encoded genes that regulate mitochondrial pathways involved in ATP generation, encoding subunits of OXPHOS and associated assembly factors, enzymes involved in ubiquinone biosynthesis, and the pyruvate dehydrogenase complex. Despite the major advances highlighted in this review, current available treatment options for mtDNA diseases are limited and focus on disease management. However, assisted reproductive techniques which allow almost complete replacement of the cytoplasm of egg/embryo, eliminating the undesired mutated mitochondria, are proposed to families with mtDNA mutations that are transmitted down the maternal lineage. The development of techniques that genetically manipulate the detrimental mtDNA sequences either in vitro or in vivo may provide potential treatment for mtDNA diseases.


The finding of hematological malignancies within bone marrow in heterotopic ossification has been reported only a handful of times previously in the literature. We described a case of true trilineage hematopoiesis in an excised area of heterotopic ossification from an enucleated blind painful eye.A 70-year-old Caucasian man, positive for asymptomatic lymphoplasmacytic lymphoma, presented with a blind painful right eye in our ophthalmology department to evaluate enucleation bulbi. After enucleation, a histopathologic examination revealed the presence of intertrabecular infiltration in the metaplastic bone marrow of non-Hodgkin B lymphoma, with small lymphocytes, with similar characteristics to the already known disease.This uncommon case reveals the possibility of the localization of malignant cells within bone metaplasia of intraocular ossification in an enucleated blind painful eye. From a practical point of view, a careful systematic histopathological examination of all resected tissues in patients with a history of malignant neoplastic pathology is necessary to confirm the diagnosis and exclude occult malignancies. The presence of bone marrow within heterotopic ossification (HO) has been reported only a few times in the literature, and reports of finding hematological malignancies within the same bone marrow are even rarer. This uncommon case reveals the possibility of localization of malignant cells within bone metaplasia and we want to highlight the importance of a systematic histopathological examination of all resected tissue in patients with a history of malignant neoplastic pathology.We report a case of true trilineage hematopoiesis in an excised area of HO from an enucleated blind painful eye.A 70-year-old Caucasian man presented with a blind painful right eye to our ophthalmology department to evaluate enucleation bulbi. He had a history of a blunt trauma injury to his right eye that occurred 49 years ago, and he subsequently experienced hyphema, vitreous hemorrhages, and traumatic cataract. Over the past 10 years, he experienced episodes of ocular pain phthisis bulbi associated with trigeminal neuralgia. A slit lamp examination of his right eye showed band keratopathy and seclusion pupillae, with a brunescent traumatic cataract. A funduscopic examination of his right eye was not executable due to media opacity. His left eye was normal without any kind of lesions and had an uncorrected visual acuity (UCVA) of 20/20. A right eye ultrasound B-scan examination showed a phthisic eye, with complete retinal detachment, and hyperechogenic tissues with an acoustic shadowing due to calcification projected into the orbital tissue. For that reason, the clinicians decided to perform the enucleation of his right eye. His past medical history was positive for asymptomatic lymphoplasmacytic lymphoma associated with immunoglobulin M (IgM) monoclonal peak diagnosed 12 years before enucleation. At the onset, neither lymphadenopathies nor hepatosplenomegaly were described, and 20% bone marrow infiltration was documented. Our patient was periodically followed up without any treatment with evidence of a slow progressive disease, which did not require treatment up until now. Prior to the enucleation, magnetic resonance imaging (MRI) of his brain was performed to study a possible trigeminal neuralgia and was normal without any cerebral lesions.The eyeball was removed and marked fibrosis with places of osseous metamorphism was noticed macroscopically. A histopathologic examination revealed diffusive fibrotic and calcified uveo-scleral flaps, with widespread bone metaplasia with hematopoietic marrow, mixed with retinal fragments with atrophic areas alternating with hyperplastic aspects and blood clots Fig. a.Fig. 1A further typing showed the presence of intertrabecular infiltration in the metaplastic bone marrow of non-Hodgkin B lymphoma, with small lymphocytes, with similar characteristics to the already known disease. Immunostaining was positive for CD20, CD3, CD10, CD43, CD23, CD138, S100 GFAP, MelanA, and Ki-67  is a non-Hodgkin lymphoma affecting the B lymphocytes of the immune system . SLL oftThis rare case reveals the possibility of localization of malignant cells in true trilineage hematopoietic tissue within HO in an enucleated blind painful eye. In managing a painful blind eye, we must subject all enucleated specimens to a systematic histopathological examination, to confirm the diagnosis and exclude a hidden intraocular malignancy.