With the increasing prevalence of diabetes worldwide, vascular complications of diabetes are also on the rise. Diabetes results in an increased risk of macrovascular complications, with atherosclerotic cardiovascular disease (CVD) being the leading cause of death in adults with diabetes. The exact mechanisms for how diabetes promotes CVD risk are still unclear, although it is evident that monocytes and macrophages are key players in all stages of atherosclerosis both in the absence and presence of diabetes, and that phenotypes of these cells are altered by the diabetic environment. Evidence suggests that at least five pro-atherogenic mechanisms involving monocytes and macrophages contribute to the accelerated atherosclerotic lesion progression and hampered lesion regression associated with diabetes. These changes include (1) increased monocyte recruitment to lesions; (2) increased inflammatory activation; (3) altered macrophage lipid accumulation and metabolism; (4) increased macrophage cell death; and (5) reduced efferocytosis. Monocyte and macrophage phenotypes and mechanisms have been revealed mostly by different animal models of diabetes. The roles of specific changes in monocytes and macrophages in humans with diabetes remain largely unknown. There is an ongoing debate on whether the changes in monocytes and macrophages are caused by altered glucose levels, insulin deficiency or insulin resistance, lipid abnormalities, or combinations of these factors. Current research in humans and mouse models suggests that reduced clearance of triglyceride-rich lipoproteins and their remnants is one important mechanism whereby diabetes adversely affects macrophages and promotes atherosclerosis and CVD risk. Although monocytes and macrophages readily respond to the diabetic environment and can be seen as protagonists in diabetes-accelerated atherosclerosis, they are likely not instigators of the increased CVD risk. To date, almost 10% of the US population has type 1 or type 2 diabetes mellitus (T1DM or T2DM), and the prevalence of diabetes continues to rise . The preBoth forms of diabetes significantly increase the risk of atherosclerotic cardiovascular disease (CVD). Atherosclerotic lesion morphology appears to be similar in people with T1DM and T2DM based on post-mortem studies, and both forms of diabetes increase atherosclerosis disease burden, enlarge lesion necrotic cores and increase lesional macrophage content (based on CD68 immunoreactivity), as compared with lesions from subjects without diabetes .Improved glycemic control using intensive insulin therapy reduces mortality and the risk of CVD in people with T1DM  even aftThe observed dyslipidemia associated with diabetes is characterized, not primarily by elevated low-density lipoprotein (LDL) cholesterol levels, but rather by elevated triglyceride-rich lipoproteins (TRLs)—very low-density lipoproteins (VLDL), chylomicrons, and their remnant lipoprotein particles (RLPs)—and by lower high-density lipoprotein (HDL) cholesterol and smaller denser LDL particles. This lipid profile, commonly referred to as diabetic dyslipidemia , is mostFurthermore, recent studies suggest that apolipoprotein C3 (APOC3), an apolipoprotein that prevents clearance of TRLs and their RLPs –18, predThe metabolic hallmarks of diabetes discussed above affect multiple cell types in the artery wall, promoting pro-atherogenic processes and progression of atherosclerosis. In particular, diabetes results in changes in the myeloid cell compartment, which includes monocytes and macrophages. In this review, we will discuss diabetes-induced alterations in monocyte and macrophage phenotypes and how these alterations might contribute to the increased CVD burden in diabetes.Monocytes and macrophages play essential roles in all stages of atherosclerosis , both inEarly studies of the effects of diabetes on macrophages and other immune cells were pursued in part because of the interest in pancreatic immune cells contributing to diabetes development. Some of these studies suggested that diabetes suppresses macrophage functions, such as phagocytosis  and migrBoth mouse and human studies implicate macrophages as a key cell type in atherosclerosis associated with diabetes –37. ThusElevated levels of circulating blood monocytes (monocytosis) can result in increased monocyte recruitment to lesions, thereby accelerating lesion progression, and can also impede atherosclerosis regression. Numerous animal studies support the idea that increased monocyte levels in circulation can result in increased monocyte recruitment to the artery wall –54. HypeTogether, these studies demonstrate that monocytosis is associated with exacerbated atherosclerosis in diabetic mouse models and that the damage-associated molecular pattern molecules S100A8 and S100A9 play a critical role in this process. Similar mechanisms might be relevant in humans because plasma S100A8/S100A9 levels were shown to correlate with leukocyte counts and coronary artery disease in patients with T1DM  and to bHowever, diabetes is not consistently associated with monocytosis in mouse models, and monocytosis is not routinely shown to be present in people with diabetes. Furthermore, in a recent study, SGLT2 inhibition accelerated atherosclerosis regression in diabetic mice without altering circulating monocyte levels , demonstTogether, these studies suggest that diabetes-induced monocytosis can contribute to, but is not necessary for, diabetes-accelerated atherosclerosis in mouse models. Thus, additional mechanisms are important in driving diabetes-accelerated atherosclerosis both in mouse models and in humans.Monocytes enter peripheral tissues in response to tissue changes the body perceives as an injury, such as atherosclerosis. Once in tissues, these cells differentiate into macrophages. Macrophages are highly plastic cells that take on different phenotypes depending on their surrounding micro-environment. Their primary roles are to phagocytose pathogens and dead and dying cells, which contributes to healing of the perceived injury, and to produce chemokines and cytokines to call in additional leukocytes to aid in the protection of the organism when needed. Macrophages thereby contribute to both the pro-inflammatory and the pro-resolution phases of inflammation.Tnfa mRNA , and IL-6, as compared to monocytes isolated from control subjects –66. Likenfa mRNA . In addinfa mRNA –65, 67. nfa mRNA .ex vivo stimulation  imply that diabetes impairs the resolution of the inflammatory response during wound healing, with macrophages from diabetic mice continuing to produce pro-inflammatory markers even during the resolution phase , 69. At e lesion . This pae lesion .in vitro, monocyte-derived macrophages from subjects with diabetes express elevated levels of numerous pro-inflammatory cytokines, including IL1B, CCL2, and IL6, as compared with monocyte-derived macrophages from controls  indicates a causative role for IL-1β in atherosclerotic cardiovascular disease in humans . SubjectA very recent single-cell RNA-sequencing study describes the transcriptome of leukocytes from asymptomatic and symptomatic human carotid atherosclerotic lesions. Strikingly, the authors found that a TLR4 and IL-1β signaling signature was associated with macrophages from asymptomatic lesions, rather than symptomatic lesions . InsteadIn summary, there is evidence that diabetes promotes a more inflammatory monocyte and macrophage phenotype, and that the heightened inflammatory capacity of macrophages might be facilitated by priming of blood monocytes or potentially of myeloid progenitor cells by the diabetic environment . It is nMacrophages in lesions of atherosclerosis will engulf APOB-containing lipoprotein particles , resulting in the formation of the hallmark cell type of atherosclerosis—foam cells. Large TRLs, such as chylomicrons derived from the gut after a meal, are believed to be too large to enter the artery wall without first being partly hydrolyzed to RLPs by lipoprotein lipase. While LDL needs to be modified, e.g., by oxidation, to be effectively ingested by macrophages, VLDLs and RLPs are readily taken up by macrophages without modification , 80. Earin vivo, the increased macrophage cholesteryl ester loading associated with diabetes was prevented while the heightened Il1b and Tnfa gene expression was unaffected —and an activating step resulting in the assembly and activation of caspase 1 and subsequent processing of pro-IL-1β and pro-IL-18 to mature IL-1β and IL-18  is required for macrophage inflammatory activation . Loss ofDisruption of fatty acid storage in inert triacylglycerol is associated with saturated fatty acid-induced inflammatory changes in macrophages . HoweverTogether, these studies indicate that aberrant lipid accumulation and handling in macrophages can cause, or is associated with, increased inflammation. Prevention of macrophage lipid loading in diabetic mice associates with prevention of accelerated atherosclerosis . FurtherHuman imaging studies suggest that diabetes accelerates lesion progression. Thus, Won et al. used coronary computed tomography angiography to demonstrate that atherosclerotic lesion progression is faster in people with diabetes than in those without, despite having lower LDL cholesterol at baseline . In a siAs the atherosclerotic lesion progresses, a necrotic core is formed. Necrotic core expansion is thought to be due, at least in part, to the death and reduced clearance of dead and dying macrophages . Both huThe clearance of dying or dead cells by phagocytes is termed efferocytosis. Efferocytosis has been shown to be impaired in mouse models of T2DM, an effect ascribed to the increased membrane content of saturated fatty acids . Reducedm for glucose of 1–7 mM, it is generally believed to be nearly saturated at physiological ranges of glucose . Several lines of evidence suggest that the presence of microvascular disease dramatically increases the risk of macrovascular complications –142. WheDo monocytes and macrophages play a causative role in DKD? Macrophages are known to accumulate in the glomerulus and renal interstitium in kidney disease –145. MonLipids accumulate in glomeruli in DKD , consistIn 2008, the Food and Drug Administration recommended to include cardiovascular endpoints in all phase 2 and phase 3 clinical trials of new anti-diabetic therapies. As a result of this recommendation, new anti-diabetic therapeutics were revealed to have unexpected protective effects on cardiovascular outcomes. Glucagon-like peptide 1 (GLP-1) receptor agonists, which increase insulin secretion, and SGLT2 inhibitors, which lower blood glucose levels by preventing glucose reabsorption in the kidney, both were shown to reduce cardiovascular events in patients with T2DM –160. GLPDo these drugs have direct effects on monocytes and macrophages and is it possible that some of the beneficial effects are due to alterations of lesion macrophages? SGLT2 inhibitors have been shown to prevent macrophage foam cell formation in diabetic mice concomitant with reduced atherosclerosis . The mecGLP-1 receptor agonists prevent incident atherosclerotic CVD in people with diabetes. Some of the effects might be mediated by direct effects on macrophages, although GLP-1 receptor agonists exert effects in a plethora of cell types, and macrophages are unlikely to be a major point of action. Nevertheless, exendin-4 (a GLP-1 receptor agonist) has been shown to reduce atherosclerosis and monocyte adhesion to aortas as well as inflammatory gene expression in macrophages in mice, likely by increasing cAMP levels in these cells .In relation to the beneficial effects of icosapent ethyl ester on incident CVD, fish oils have been shown to have beneficial effects in macrophages, including reversal of the defective efferocytosis associated with diabetes . AlthougMore research is needed to understand the beneficial effects of these therapies in patients with diabetes. Such studies could also shed additional light on the mechanisms whereby diabetes increases CVD risk.Diabetes creates an environment that fosters the initiation and progression of the atherosclerotic lesion as well as a hampered lesion regression through at least five likely mechanisms in monocytes and macrophages . First, Many pieces of this puzzle remain to be found. Although monocytes and macrophages are present in all stages of atherosclerosis and their function is altered by diabetes, a major question is whether the myeloid cell compartment could be successfully targeted for prevention of diabetes-accelerated atherosclerosis and CVD. The CANTOS trial was the first trial to target inflammation and produce a protective effect on CVD. Such an approach might be feasible and successful in people with diabetes and heightened inflammation. Furthermore, based on the research available to date, new therapies that target apolipoproteins, such as APOC3, would be predicted to be effective in preventing CVD in patients with diabetes and elevated levels of APOC3. Other important questions for future research include the mechanisms whereby diabetes induces adverse effects in myeloid cells. For example, what exactly goes awry in myeloid cell lipid handling in the setting of diabetes, and what is the mechanism whereby lipids accumulate in these cells? What is the role of inflammatory changes vis-à-vis lipid over-accumulation? What changes are critical for necrotic core expansion and CVD events? Do different myeloid cell populations respond differently to the diabetic environment? Some of these important answers are likely to be revealed by new methodology, such as single-cell RNA-sequencing, proteomics, metabolomics and lipidomics.JK and C-CH wrote the manuscript. KB wrote parts of the manuscript and edited the full manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Extracorporeal shock wave therapy (ESWT) is a non-invasive therapeutic method used for pain management and muscle strength improvement through the use of shock waves. In vitro studies have demonstrated that shockwave therapy induces fluctuation in redox reaction regulation and increases in Mitogen-Activated Protein Kinase (MAPK) signal transduction pathways, stimulating increased gene expression in the nucleus. ESWT has also been shown to upregulate angiogenesis and growth factors through activation of endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF). The use of ESWT in the treatment of various musculoskeletal disorders was widely adopted throughout Europe, South America, and Asia before being introduced in the United States in 2000. Within the past 20 years, the clinical application of ESWT in the treatment of musculoskeletal and bone disorders has grown. This paper provides a comprehensive narrative review of applications and outcomes of ESWT in clinical spinal pathology and assesses reported efficacy as it relates to the pathology. A review of the literature yielded studies describing the use of ESWT in degenerative osteoporotic neuro-spinal pathology, heterotopic ossification due to spinal cord injury, cervical spondylosis, scoliosis, sacroiliitis, and coccydynia. The efficacy of ESWT as an adjunct treatment in patients with spinal cord pathologies varied with the specific pathology, however, all pathologies discussed in this review provided evidence of potential benefits with minimal adverse effects. While the use of ESWT for pain management has widely been established, further literature should aim to identify the long-term benefits of ESWT. Extracorporeal shock wave therapy (ESWT) is a non-invasive therapeutic method used for pain management and muscle strength improvement through the use of shock waves to promote revascularization . The conWhile the potential of ESWT in the treatment of a wide variety of conditions has been acknowledged, current FDA approved indications of use remain limited, suggesting the need for further research discussing the efficacy of ESWT in alternative indications. The objective of this paper is to provide a comprehensive review of potential indications for ESWT in spinal cord pathology and assess efficacy as it relates to the individual pathology. Efficacy will be determined through the analysis of outcomes from currently published studies on ESWT as a treatment protocol in degenerative osteoporotic neuro-spinal pathology, heterotopic ossification due to spinal cord injury, cervical spondylosis, scoliosis, sacroiliitis, and coccydynia.Mechanism of ESWT therapyExtracorporeal shockwaves are rapid oscillations of pressure waves that can travel through various mediums. A shockwave is a non-linear pressure with a duration of 10 microseconds. There is a positive phase, characterized by high-pressure waves hitting a tissue interface that allow penetration or reflection. During the negative phase, the accelerated pressure causes the tissue layers to cavitate, inducing an air bubble formation and produces a second shock wave . The eneExtracorporeal shockwave therapy is divided into focused shockwave therapy (FSWT) and radial shockwave therapy (RSWT). In focused shockwave therapy, a pressure field converges into a selected focus frame at specific depths within body tissue. There are three different shockwaves used, including electrohydraulic, electromagnetic, and piezoelectric which are all waves generated in water . Water aApplicationsOsteoporosisOsteoporosis commonly affects the elderly and post-menopausal women and has multiple clinical presentations in the spine, most commonly compression fractures of the vertebral body and resultant radiculopathies . While tShi et al. designed a randomized control trial (RTC) of 64 postmenopausal patients with a known history of osteoporosis with two treatment groups n=20,21) and one control (n=23). Treatment groups were low energy flux density ESWT  and high energy flux density ESWT . These were applied by a Dornier Compact Delta II Emitter  with penetration depth and focus diameter of 15 and 4 mm, respectively  under ultrasound guidance. Application of mild energy flux density ESWT  was administered seven times weekly for seven weeks. Although the size of NHO measured with radiograph was not reduced significantly, the patient’s VAS score was reduced to a 3, with the therapeutic effects lasting for six months. Li et al. described ESWT in a patient with a history significant for incomplete C8 SCIaccording to American Spinal Injury Association (ASIA) Impairment Scale D with reported pain VAS of 8 in the left hip [Jeon et al. reported application of ESWT in a wheelchair-bound patient with complete C4 SCI according to American Spinal Injury Association (ASIA) Impairment Scale A with reported pain Visual Analog Scale VAS, 0-10) of 8 in the right hip . Followi of 8 in Cervical SpondylosisThe nuchal ligament is an important structure that maintains lordotic alignment and stabilizes the head and cervical spine during movement , 28. OssIn this study, prior to treatment, cross-sectional area (CSA) of ONL was measured with x-ray radiograph, range of motion(ROM) was measured with a goniometer, neck disability was measured with neck disability index  and pain was measured with VAS (0-10). All three study groups received hot packs and traction force to the cervical spine, while the two treatment groups received additional x-ray guided ESWT or musculoskeletal sonography guided ESWT. In both treatment groups, ESWT was applied using an F10G4 shockwave generator   to the patient’s flexed neck in a seated position, once weekly for six weeks. At three months following complement of treatment, ESWT groups showed significant improvement of NDI, VAS, and ROM over both the initial assessment and the conservative treatment control group (hot packs/traction). Throughout each of these outcome measures, musculoskeletal sonography guided ESWT had more favorable results compared with X-ray guided, showing a significant improvement over x-ray guided ESWT specifically in neck bending ROM. CSA was the only outcome measure that did not significantly change in any of the groups throughout the course of treatment or at follow up. This study demonstrated ESWT as an effective adjuvant treatment to achieve pain reduction, improved ROM, and lower NDI scores in patients with cervical spondylosis and calcification of the nuchal ligament.ScoliosisThe first use of ESWT for the treatment of scoliosis was described by Weiss et al. in 2013 through a pilot study of 15 adolescent patients . The ratThus, in the ESWT pilot study for scoliosis, therapy was localized to the lower thoracic segments where the “flatback contracture” was present and theoretically, where ESWT treatment was most likely to correct IFF and improve overall 3D deformity of the spine caused by tethering. A one-time ESWT treatment was administered to 15 girls with scoliosis  with a Storz Medical Masterpuls MP 100 system  with trigger point applicator . Outcomes were measured five minutes post-treatment and demonstrated consistent reduction of lateral deviation and surface rotation (desirable) as well as increased kyphosis and lordosis angles (desirable) when compared to pre-treatment values. Although effects were no longer present within a few days, Weiss et al. concluded that vertebral improvements post-intervention fit into the concept of functional tethering. The idea being that ESWT temporally improved spinal restriction that had occurred chronically over time due to continuous functional tethering on the spine . In 2017SacroiliitisThe sacroiliac joint is a diarthrodial joint that connects the axial skeleton to the lower extremities . DisruptMoon et al. conducted a randomized sham-controlled trial that consisted of 25 patients with SIJ pain referred to the rehabilitation clinic at a university hospital . The parThe intensity of pain and understanding of function was evaluated using the numeric rating scale (NRS) and Oswestry disability index (ODI), respectively, both prior to treatment and four weeks after. At four weeks post-treatment, NRS scores in the ESWT group showed significant improvement compared to baseline with no significant improvement observed in the control group. The ODI scores in contrast, did not show any significant post-treatment changes in both groups. However, the ODI score in the ESWT group did exhibit a notable trend towards improvement at both one and four weeks post-treatment as compared to baseline. Finally, there was a significant difference between the NRS scores in the ESWT and control group at four weeks post-treatment but no significant difference in ODI scores. It is hypothesized that ESWT improves pain by either damaging, and thus suppressing pain-conducting unmyelinated fibers, and/or by inducing neovascularization and tissue regeneration of the ligamentous structure that forms the posterior aspect of the SIJ -44. ThusCoccydyniaCoccydynia occurs due to inflammation of the coccyx, or its joint with the sacral vertebra, causing pain most commonly experienced upon moving from a seated to standing position , 45. TheAnalysis of current published studies suggests that Marwan et al. was the first to describe the use of ESWT for coccydynia, reporting favorable results in treating two patients . Over thThe use of ESWT in patients with a known history of osteoporosis may be indicated, as suggested by the increased BMD levels and interpretation of decreased degenerative changes in the 12-month intervention following treatment. ESWT for the treatment of pain secondary to heterotopic ossification due to spinal cord injury was found to be effective through continued use of ESWT, as indicated by decreased VAS scores reported by patients and decreased NHO measurements. Similarly, the treatment of pain secondary to cervical spondylosis also suggested the effectiveness of ESWT, as indicated by decreased patient-reported pain levels. Furthermore, the use of ESWT as an adjunct treatment in patients with cervical spondylosis and calcification of the nuchal ligament was associated with improved ROM and lower NDI scores, suggesting the benefits of ESWT are not limited to pain reduction. Despite initial improvements as indicated by improved finger to floor distance (FFD) 22.6cm to 15.6cm (p <0.01) and angle of trunk rotation (ATR) 3 degrees to 10 degrees (p <0.05) following ESWT in patients with scoliosis, the long-term benefits of ESWT for patients with scoliosis was not established due to consistent benefit reversal three to five days post-intervention. In patients with sacroiliitis, significant improvements in SIJ pain and the absence of patient reported side effects following ESWT intervention indicate ESWT may be considered for the treatment of SIJ pain, however further research is warranted due to limited sample size and lack of long-term follow up. Despite decreased VAS pain scores and subjective satisfaction in patients with coccydynia following ESWT treatment, further research is warranted to establish the long term benefits of use due to findings suggesting the reversal of the effects of ESWT and limitations of the studies, including a lack of a control group.The efficacy of ESWT as an adjunct treatment in patients with spinal cord pathologies varied with the specific pathology, however, all pathologies discussed in this review provided evidence of potential benefits with minimal adverse effects. While the use of ESWT for pain management has widely been established, further literature should aim to identify alternative benefits, including the role of ESWT in bone healing, and to establish the long-term benefits of ESWT.


Catellicoccus associated with seabirds. MST assessments were combined with local knowledge, assessments of sanitary infrastructure, and routine watershed surveys. This study identified hotspots of human FIB along the western Saipan Lagoon shoreline in both surface waters and groundwater, plus another hotspot of human FIB at a popular tourist bathing area known as the Grotto. FIB hotspots on the Lagoon shoreline coincided with areas of high SGD and nitrogen isotopic data indicating sewage-derived N inputs. It appears that faulty sanitary infrastructure may be contributing to inputs to Saipan Lagoon, while bather shedding is likely a primary input for the Grotto area. Moderate levels of dog fecal contamination were common and widespread across the island. High levels of seabird fecal contamination were more random, both spatially and temporally, and mostly concentrated along the less developed northeast region of Saipan. No significant levels of cow or pig fecal marker were detected in coastal water samples. This study provides demonstration and establishment of analytical capacity to resource management in CNMI for MST technology to aid in trouble-shooting water quality issues involving land-based sources of microbial contaminants to CNMI coastal waters.The Commonwealth of the Northern Mariana Islands (CNMI) recently identified the need to improve its capacity for detecting and tracking land-based sources of pollution (LBSP) in coastal waters, particularly microbial contaminants like fecal indicator bacteria (FIB). Reported here is a baseline study of a suite of host-specific FIB microbial source tracking (MST) markers in the coastal shoreline and reef waters around the island of Saipan. Three sampling campaigns were conducted in September 2017, March 2018, and August 2018. Samples were collected from the nearshore surface waters of Saipan, the reef waters of Saipan Lagoon, and groundwater from beaches along the Saipan Lagoon shoreline. Measurements of submarine groundwater discharge (SGD) into nearshore waters and isotopic source tracking of nitrogen inputs were conducted concurrently with MST. Environmental DNA was extracted from the samples and analyzed by quantitative polymerase chain reaction (qPCR) for MST gene markers of fecal Bacteroidales specifically associated with humans, dogs, cows, and pigs, and for an MST gene marker of  The Commonwealth of the Northern Mariana Islands (CNMI) is a territory of the United States that comprises all the islands of the Northern Mariana archipelago chain in the northwest Pacific, except for the island of Guam (which is its own separate US territory). Saipan, the largest of the 14 islands of the CNMI, is the capital and houses the largest population, with the majority of communities situated along Saipan’s western coast. Waters along this area are negatively impacted by chronic sediment, nutrient, and microbial pollution that, combined with other stressors such as temperature-induced bleaching, ocean acidification, fishing pressures, reef tourism pressures, etc., can affect many of the island’s western and southeastern reefs . Saipan’Management of the CNMI’s coastal resources falls under the jurisdiction of the Bureau of Environmental and Coastal Quality (BECQ). The BECQ is responsible for monitoring, assessing, and protecting water quality within the CNMI, as well as managing land, air, water, and coastal quality. Both commonwealth and US federal laws and regulations mandate this responsibility . As a whIn recent years, the BECQ has issued numerous water quality violations due to elevated enterococci fecal indicating bacteria (FIB) levels in areas of Saipan with heavy stormwater runoff . Many ofThe CNMI has 240.5 miles of ocean shoreline. In 2018, 50.5 of these coastline miles (or 21% of the CNMI’s coastline miles) were found to be impaired due to exceedance violations of enterococci FIB levels . 32.7 miEscherichia coli with the ColiLert system (IDEXX Laboratories) as per the manufacturer’s instructions and guidelines by the US Environmental Protection Agency (EPA). However, these current regulatory approved tests lack any source-tracking capability. Although general FIB monitoring is useful for identifying hotspots and zones of water quality exceedance, it is of less utility in helping resource managers understand why there are such exceedances and where the microbial contamination may be originating . It also regularly tests for ginating .E. coli and enterococci FIB. The system consists of a Pall GeneDisc qPCR thermocycler and Pall Environmental DNA Extractor System used with the GeneDisc Recreation Water E. coli and Enterococcus spp. assay kit . However, these particular commercial qPCR assays are for general FIB enumeration and lack source tracking capability. This project has now adapted the in-house BECQ GeneDisc qPCR thermocycler platform for conducting a range of standard host-specific microbial source tracking (MST) assays.The BECQ already uses a GeneDisc Rapid Microbiology System and a commercially available kit  to conduct quantitative polymerase chain reaction (qPCR) enumeration of Many strains and species of gut microbial flora have co-evolved with their animal hosts and contain unique gene sequences that can be diagnostic for particular host-associated FIBs, indicating fecal contamination specific to particular host animals such as humans, dogs, birds, pigs, cows, etc. . The abiE. coli as a troubleshooting approach to investigate chronic microbial water quality problems. This type of approach can be highly effective when integrated into a multi-tool, multi-tiered strategy for water quality assessment, as described in The California Microbial Source Identification Manual: A Tiered Approach to Identifying Fecal Pollution Sources to Beaches . These researchers concluded that the risk associated with exposure to a fixed HF183 concentration in recreational water decreased with the age of contamination, and that swimmer exposure to sewage after it had aged ∼3 days resulted in a median risk of less than 30 illnesses per 1000 bathers. This QMRA study calculated a risk-based HF183 threshold of 900 copies/100 mL for sewage contamination aged 2.5 days, while a risk-based water quality threshold for HF183 in surface waters that takes into account uncertainty in contamination age was derived to be 4100 copies/100 mL  study by s/100 mL . ConsideAs part of the broader MST technology transfer project between the NOAA-Atlantic Oceanographic and Meteorological Laboratory (AOML) and the CNMI-BECQ, an initial study was conducted as a collaborative effort between the BECQ, NOAA-AOML, and American University. This study examined land-based sources of microbial pollution to the coastal waters of Saipan following the principles and protocols of the California Microbial Source Identification Manual . The goaOur study area was the island of Saipan, bounded by north latitude 15.290533°, south latitude 15.091500°, west longitude 145.685494°, and east longitude 145.830316°. Sampling sites included Saipan Lagoon and the Saipan reef tract along the western side of the island, sites at the northern end of the island, particularly the Grotto area (site NEB01), Bird Island (site NEB02), selected beaches along the northeast portion of the island, and the Lao Lao Bay area on the eastern side of Saipan.Field sampling efforts for the Saipan MST baseline study were conducted on March 12-19, 2018 and July 31-August 8, 2018. A few samples of opportunity were collected on September 13, 2017 (before the baseline MST survey started) as part of a routine BECQ monitoring effort, and were used primarily for demonstration purposes to teach the MST qPCR methods to BECQ personnel during the first MST technology transfer workshop conducted by NOAA-AOML. During the actual MST baseline surveys in 2018, BECQ researchers collected almost half of the samples during their routine water quality surveillance efforts, while researchers at American University collected shore and reef samples as a courtesy during their own ongoing nitrogen isotope and SGD tracking studies , which oSample sites are shown in Surface water samples were collected approximately 15–30 cm below the surface in sterile 1.5 L Whirl-Pak bags (Nasco). Samples from these nearshore and reef locations were collected as per EPA methods 1611 and 1696, stored on ice, and filtered within 6 h of collection in the BECQ laboratory at Saipan , 2019. GLive enterococci were measured from subsamples of the collected water samples using the standard EPA approved EnteroLert Fluorogenic Substrate method (IDEXX Laboratories), following EPA guidelines, established BECQ protocols, and the manufacturer’s instructions. Samples were processed with the IDEXX EnteroLert method by the BECQ Environmental Surveillance Laboratory. However, not all of samples collected by the American University group were tested using EnteroLert. The viable enterococci abundance in units of Most Probable Number (MPN) per 100 mL as estimated by the EnteroLert assay from the manufacturer’s statistical tables, is shown in In brief, at the BECQ laboratory, aliquots of water samples were filtered through sterile 0.45 μm pore-size, 47 mm diameter membrane filters using disposable sterile filter funnels . Water samples were filtered to 1 L or until clogging, and the volumes filtered varied from <100 mL to 1 l, depending on turbidity . Most samples ranged from 800 mL to 1 L. In all cases, the actual sample volume that was filtered for each individual sample was recorded and used for quantitative calculations of MST target signal. Actual volumes filtered for each sample are listed in online More specific details of adapting these MST assays to the Pall GeneDisc system are described in the online Bacteroides assay HF183 (EPA Taqman version as per EPA method 1696); (2) dog-source Bacteroidales assay DogBact; (3) cow-source Bacteroidales assay CowM2; (4) pig-source Bacteroidales assay Pig2Bact; and (5) the Gull2 assay specific for Catellicoccus marimammalium. C. marimammalium are found in the gut of most seagulls, as well as potentially in the gut of other birds  that may co-habit, scavenge, or nest with seagulls. Depending on the specific geographic location and co-nesting behavior, this may also include species of terns, pelicans, geese, and very often, pigeons  human-source  pigeons . Assay p pigeons , with mi pigeons . The seqMore specific details are given in the online Statistical boxplot analyses of the MST result patterns for host-specific fecal bacterial markers were generated with the SigmaPlot software package, version 14 . Final MST positive control concentration standard curves and their associated linear regression statistics were also plotted using the SigmaPlot v.14 software package.15N relative to atmospheric N2 (δ15N = [(Rsample/RAIR)-1]∗1000). Two in-house standards, acetanilide and caffeine  were used for calibration and the determination of the precision (0.2‰). Although several species of alga were widespread along the coast, none were present at all sites. Thus, we created an integrated primary producer δ15N value using correlations between the most common species, Caulerpa, with three others to predict δ15N values for Caulerpa at sites where it was absent.Sampling of benthic seagrass, algae, surface water and groundwater for isotopic analysis are described thoroughly in 15N and δ18O were determined using the denitrifier method (2 for δ15N and V-SMOW for δ18O.Water samples were syringe-filtered (0.2 μm) into 50-mL HDPE screw-top bottles and kept in a cooler until they could be frozen at the end of each sampling day. Then, δr method  at Hong 3 m–1 d–1) was calculated based on Ra flux  and groundwater endmember Ra activity  at three sites; SGD at other sites was estimated based on the assumption that Ra, Rn, and SGD all followed the same spatial distribution along Saipan’s coast. SGD estimates are given as a range (upper and lower) to reflect sampling error and analytical uncertainty propagated through the calculations.As detailed in The summary of results of all fecal indicator bacteria measurements for this study are shown in the project data summary table in the online The BECQ reported non-detects for IDEXX EnteroLert as <30 MPN/100 mL based on the IDEXX EnteroLert statistical tables for MPN. For the live enterococci boxplots shown in For viable enterococci, 14 of the 92 EnteroLert samples (15.2%) exceeded the EPA recommended BAV threshold of ≤70 MPN/100 mL. The statistical distribution of these exceedances is shown in the last boxplot on the right side of Bacteroides qPCR marker in surface water for recent human fecal contamination (≤3 days old), while the “exposure threshold 2” line indicates our suggested bather exposure threshold for the HF183 marker in surface water for human fecal contamination of uncertain age or >3 days old. Our suggestions for these HF183 exposure thresholds used to help interpret the results of this Saipan MST study were adapted from information by a previous Quantitative Microbial Risk Assessment study of the HF183 marker . As we utilized synthetic DNA gene blocks containing the assay target sequences for quantitation standards, we report our qPCR results here as “target sequence copies” or tsc. While terminology in the current literature varies, this “tsc” unit used here is essentially equivalent to the more common term “gene copies” (GC) or “target copies” (TC) or just “copies” as used in other papers. Note that tsc does not necessarily represent a “genome equivalent” (GE) or “cell calibrator equivalent” (CCE), as genomes may have multiple target copies of a particular assay target gene per genome depending upon the target gene of the qPCR assay .Bacteroides HF183 marker, 59 samples out of 182 total samples (32.4%) demonstrated levels over 100 target sequence copies (tsc) per 100 mL, 11 samples out of 182 were over 1000 tsc/100 mL, and 8 samples out of 182 were over 4100 tsc/100 mL. This indicates a 6.0% exceedance rate for the suggested HF183 exposure threshold for recent human fecal contamination in surface waters and a 4.4% exceedance rate for the suggested exposure threshold for fecal contamination of uncertain age. These exceedances, primarily clustered along the shoreline of the western central Saipan Lagoon region, ranged from sites WB17 to S22, plus site NEB01 (the Grotto).For the human-source fecal Bacteroides FIB marker in beach groundwater samples. Groundwater site GW-S18 had 46,062 tsc/100 mL on August 3 and 30,293 tsc/100 mL on August 7, while groundwater site GW-S19 had 5,550 tsc/100 mL on August 3 and 4,814 tsc/100 mL on August 7  to 12.0‰  and were generally higher in samples from the Garapan region of the Lagoon, and among those samples collected in March  to 10.9 m3 m–1 d–1  and overall, was most prevalent in Garapan Lagoon  frequently had elevations of both live enterococci and human fecal HF183 marker, plus some elevations of seabird marker, as shown in As part of the broader NOAA technology transition effort to aid BECQ in developing an in-house capacity for qPCR-based molecular MST within the Bureau for Environmental and Coastal Quality, NOAA-AOML and CNMI-BECQ researchers conducted a joint MST baseline study to assess the patterns of host-source FIB from LBSP in the coastal waters of Saipan. This study coincided with and was supported by sampling assistance from American University, which was conducting a separately funded independent study in Saipan of nutrient measurements, nitrogen stable isotope source tracking for nutrient pollution, and tracking radon (Rn) and radium (Ra) isotopes as natural tracers of SGD .Our study found that the predominant inputs of FIB into the critical and protected Saipan Lagoon system and reef tract ecosystems were primarily from human and dog fecal sources. Birds were also a major contributor of fecal bacteria at certain sites, especially along the more sparsely settled northern and northeastern parts of Saipan. The distribution of dog fecal bacteria contamination was relatively ubiquitous around the island and was especially prevalent in the Tanapag area, the urbanized Garapan district, and along the central western coastline bordering Saipan Lagoon. In addition, this Garapan Lagoon region of the Saipan Lagoon system is subject to substantial submarine groundwater discharge (SGD), especially in the sections from sites S17 to S21, which also carry sewage-derived N into the coastal environment .Two primary hotspots with high levels of human FIB were observed: a wide stretch of the urbanized and developed central western Saipan Lagoon shoreline in the Garapan Lagoon region, and also at the Grotto, an isolated coastal grotto swimming and scuba diving area on the northeast end of the island that attracts extremely large numbers of tourists. Both areas have also been noted as chronic hotspots for elevated enterococci during routine water quality monitoring by the BECQ, although there are other chronic enterococci hotspots previously observed by the BECQ around the island where we did not observe associated high levels of the human marker during this study.Bacteroides targeted by the HF183 marker, and some experiments have shown that the source of feces can affect die-off of fecal bacteria and the environmental persistence of fecal source tracking markers . The HF183 levels observed in these samples reflect a potentially serious exposure risk to public health and may be suggestive of significant leakage of sanitary infrastructure at those sites on those sample dates. It should be noted that these extreme exceedances of the HF183 marker occurred at sites relatively close to one another (sites S18 and S20) and repeatedly over the course of a few days in the region of the western central Saipan Lagoon shoreline. Two of these extreme exceedance samples were from groundwater samples at site S18, collected on August 3 and August 7 respectively, in 2018. The third extreme level of human FIB marker was observed in the shoreline sample of site S20 collected on August 2, 2018. S18 was consistently the site with the highest SGD, and S20 was also located within the high-SGD area of Garapan Lagoon. Additionally, SGD across all sites was higher in August (rainy season). This suggests that SGD may transport dangerous levels of human fecal pollution to the coast. All shore sites in this region of the Saipan Lagoon had elevated HF183 marker levels and accounted for the majority of the exceedances of the QMRA exposure thresholds for our data set (with the other primary exceedance site for HF183 being at the Grotto). The urban area of west central Saipan has substantial aging sanitary infrastructure, and it is probable that the observed elevations in human fecal marker were associated with compromised and leaking sanitary infrastructure sources in the area. Indeed, following the conclusion of this study, some compromised sanitary infrastructure in the region of highest human fecal markers (particularly in the groundwater) was later discovered and addressed for mitigation.This region of the west central Saipan Lagoon shoreline also had elevated dog fecal bacterial marker in many of the samples. Dog fecal bacterial marker appeared to be relatively widespread about the island, with many sample sites having significant elevations, including the Tanapag Meeting Hall area, the GrandVrio and Fiesta hotels, the Central Repair Shop area, the Garapan Fishing Dock area, and throughout the western central region of the Saipan Lagoon shoreline. Dog fecal contamination has been shown to have a substantial impact on microbial water quality . Althoug15N and SGD observations from this study have been separately published in more detail elsewhere  and were the most pronounced near site S18. When surface and groundwater were analyzed for nutrients, groundwater nitrate concentrations were nearly an order of magnitude higher than those in surface water, indicating groundwater flow was an important pathway of nitrogen pollution into Saipan Lagoon at the time of the study.The results of the nitrogen stable isotope nutrient tracking and Rn-derived SGD measurements from the Tanapag, Garapan, and Chalan Kanoa regions of the western Saipan Lagoon are generally consistent with the qPCR MST observations from this area. The δlsewhere . In summhis area , but humThese observations of nutrients, stable nitrogen isotopes, and the natural SGD tracer Rn were consistent with observations of the patterns of human FIB input into this same area of Saipan Lagoon. Our MST study along with the stable nitrogen isotope and SGD study collectively observed the groundwater and shoreline samples from this area for sites S18–S22 to simultaneously have elevated human-source fecal bacteria, elevated nutrients, and elevated Rn, strongly implicating contaminated groundwater input to the Saipan Lagoon in this area. The greatest chronic export of high levels of FIB, and particularly high-risk human-source FIB appeared to come from groundwater discharge and shoreline runoff discharge along the western central Saipan Lagoon region stretching from roughly the Garapan Fishing Dock to Susupe, with the worst of the discharge in areas near sites S18 through S20. This is likely the LBSP microbial contaminant source causing the greatest risk exposure of LBSP pathogens to the coral reefs of Saipan Lagoon (at least at the time of our study). Reef waters just offshore from this area also showed the greatest abundance of LBSP microbial contaminants, particularly human FIB marker.The other hotspot for the HF183 human fecal marker was at the Grotto, site NEB01, a popular tourist destination lacking sufficient sanitary infrastructure. There is only one restroom facility near a parking lot at the top of a cliff located a distance from the Grotto. The Grotto is protected from open ocean wave action, but is tidally flushed. It often experiences high densities of swimmers, bathers, scuba divers, and general tourists who frequently visit this natural attraction in large groups. The swimming areas of the Grotto can only be accessed by descending a very long, steep stairway cut into the cliffside. Large crowds often move up and down the stairway, making transit into and out of the Grotto area difficult and time consuming. There are no restroom facilities in the Grotto area itself, and due to the challenging climb up the stairway, many visiting swimmers may either be unable or unwilling to access the restroom in the parking area at the top of the cliff.Enterococcus, fecal Bacteroides, and Staphylococcus aureus among others . However, these later observations from the Grotto about the reductions in enterococci levels during the COVID-19 pandemic and beach closures are consistent with the human-marker MST data presented here, and further support the idea that bather shedding is a primary input of FIBs in the Grotto area.E. coli, especially as bird fecal contributions may serve to confound general water quality assessments. If significant bird contribution is determined to be a primary contributing factor in recreational water sites of chronic exceedance without any other high-risk fecal input sources, alternative site-specific water quality criteria might be considered. In this case, a combination of methods, including MST and Quantitative Microbial Risk Assessment, could be deployed to help confirm if any such alternative criteria promulgated by the BECQ would likely be equally protective of both public and environmental health.Bird fecal input appeared to be a major contributor to other observed enterococci hotspots, with areas such as Bird Island and American Memorial Park Drainage routinely having regulatory enterococci exceedances and high levels of Gull2 seabird marker. No other significant sources of fecal marker were found in the water samples from Bird Island; it is therefore logical to assume that bird populations are the primary cause for the chronic enterococci water quality failures of this area. Based on our Gull2 qPCR results, birds also appeared to be major fecal contributors to other areas of chronic enterococci exceedance, such as Jeffrey’s Beach and Old Man by the Sea. Thus, birds would appear to be significant contributors of FIB at areas such as the American Memorial Park Drainage, the Lao Lao Bay area, the Grotto, Jeffrey’s Beach, Old Man by the Sea and, of course, Bird Island. Such bird fecal contamination is probably not manageable in a practical sense. However, the potential of bird fecal contributions and their relatively lower risk, should be considered when assessing observed exceedances of general fecal indicators such as enterococci or In examining coral reef exposure, this study found that LBSP-derived microbial contaminants were being transported from the Saipan Lagoon coastline to the reef tract. Therefore, the reefs offshore from the Saipan Lagoon coast may also potentially be exposed to pathogens and other contaminants associated with this LBSP pollutant transport . While reef waters were tested during this study, coral tissues were not. Consequently, it could not be determined whether microbial contaminants were being absorbed into or influencing the coral holobionts at these reefs. However, impacts of LBSP microbial contaminants on coral microbiota have been documented at other coral reefs, including reefs in southeast Florida . A folloBased on this MST study, the following recommendations have been made to the resource managers of the CNMI-BECQ:(1)Conduct further investigations of sanitary infrastructure and potential leakages , especially along the west central Saipan Lagoon shoreline, and aggressively pursue the detection and repair of sanitary infrastructure in the region.(2)Conduct further investigations of the potential contamination of stormwater runoff, especially in the regions of Tanapag, Garapan district , Susupe, and the west central Saipan Lagoon shoreline.(3)Implement more robust control measures for dealing with animal waste, particularly dog fecal contamination, especially along the western coast of Saipan Lagoon. A large number of dog fecal marker elevations suggest that dog fecal influence is widespread. Among other things, consider mandating dog fecal cleanup policies and provide the necessary resources to make dog fecal cleanup in public spaces easy and cost effective.(4)Implement more robust bather crowd control measures at high density bathing beaches and recreational water areas, especially at the Grotto, along with more accurate bather density monitoring of crowds at the Grotto. Managing crowd size at the Grotto will allow more time for natural tidal flushing, which might be advantageous to maintaining better water quality at the site. It would also likely provide a benefit in increased physical safety and reduced accidents.(5)Integrate periodic molecular MST assessments into the more routine water quality monitoring efforts of the BECQ to permit a better understanding of the seasonality, infrastructure, environmental, and social factors that influence source contributions of contaminants to local waters, and to help trouble-shoot any sites of chronic exceedances. BECQ is now also planning to expand MST assessments to other less populated islands of CNMI, including Tinian and Rota, where it is suspected that the predominant FIB sources may be dogs and birds, rather than humans as in Saipan .(6)Further test the CowM2 and Pig2Bact qPCR assays to determine whether local or regional factors  affect the detection sensitivity of these two livestock assays. If this turns out to be the case, modified or alternate MST protocols for livestock could be developed or adopted to boost the detection sensitivity for local Saipan livestock fecal contamination. Also, supplement testing with the use of a more general ruminant fecal marker such as the Rum2Bact assay.The results from this study demonstrate the value of integrating molecular MST technology into the assessments of water quality in the CNMI, along with providing the first baseline assessment of host-specific fecal water quality indicators for the CNMI region.In conclusion, we identified a need for molecular MST capability within CNMI regulatory agencies. We initiated an MST technology transition effort between NOAA-AOML and the CNMI-BECQ and used this new MST capacity at the BECQ to conduct the first baseline assessment of host-specific fecal water quality microbial indicators in the coastal waters and protected coral reefs of Saipan. We found that the west central shoreline region of Saipan Lagoon was the greatest contributor of human-specific fecal bacteria contamination to the lagoon and reef tract during the period of study. Our study also identified another hotspot of human fecal marker contamination at the popular recreational swimming and scuba diving site of the Grotto. We have suggested that the primary input of fecal bacteria for the Saipan Lagoon is most likely compromised sanitary infrastructure along the western Saipan Lagoon shoreline from Garapan Fishing Dock to Susupe, while at the Grotto the most likely cause is the high density of human bathers.Birds also appeared to be significant contributors of FIB to areas such as the American Memorial Park Drainage, the Lao Lao Bay area, the Grotto, Jeffrey’s Beach, Old Man by the Sea, and, of course, Bird Island. Such bird fecal input is probably not manageable in a practical sense, but it should be recognized that bird fecal contributions may serve to confound general water quality risk assessments such as the use of general culture-based enterococci tests. We also detected much less fecal marker associated with pigs and cows than expected, but we suspect the particular qPCR assays used (CowM2 and Pig2Bact) might have a lower sensitivity to the specific populations of Saipan livestock. We recommend that further investigation and optimization of MST methods for these specific populations of Saipan livestock may be warranted, as well as use of more general ruminant fecal qPCR assays. This study also found that moderate levels of dog fecal contamination were widespread on the island and have suggested a more robust control of animal fecal waste management. Overall, we believe the inclusion of molecular MST capability as demonstrated here can be a useful addition to the water quality toolbox of resource managers in the CNMI to enhance the ability of the BECQ to better protect public health and that of the environment.The original contributions presented in the study are included in the article/CS conceived and designed the experiments, performed the experiments, contributed reagents, materials, and analysis tools, analyzed the data, prepared the figures and/or tables, authored or reviewed drafts of manuscript, approved the final draft, and serves as Principal Investigator and as corresponding author. KK conceived and designed the stable nitrogen isotope experiments, participated in sampling, analyzed the data, prepared the figures, authored or reviewed drafts of manuscript, and approved the final draft. MG conceived and designed the experiments, performed the experiments, contributed reagents, materials, and analysis tools, analyzed the data, prepared the figures and/or tables, authored or reviewed drafts of manuscript, and approved the final draft. KY conceived and designed the experiments, analyzed the data, prepared the figures and/or tables, authored or reviewed drafts of manuscript, and approved the final draft. KKn conceived and designed the SGD experiments, participated in sampling, analyzed the data, authored or reviewed drafts of manuscript, and approved the final draft. DP performed the experiments, contributed reagents, materials, and analysis tools, reviewed drafts of manuscript, and approved the final draft. CB performed the experiments, contributed reagents, materials, and analysis tools, authored or reviewed drafts of manuscript, and approved the final draft. AB performed the experiments, analyzed the data, reviewed drafts of manuscript, and approved the final draft. HL performed the experiments, analyzed the data, reviewed drafts of manuscript, and approved the final draft. LM provided oversight for BECQ activities for the project and the manuscript, contributed materials and analysis tools, reviewed drafts of manuscript, and approved the final draft.Neither the United States Government nor any of its employees, contractors, or their employees make any warranty, expressed or implied, or assumes any legal liability or responsibility for any third party’s use of apparatus, product, or process discussed in this document, or represents that its use by such party would not infringe on privately owned rights. Mention of any commercial entities, trade names, or commercial products in this document does not constitute any type of endorsement or recommendation for use.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To meet the incredible demand of MRI, ultra-high-field T2 MRI is gradually attracting the attention of research and medical needs owing to its high resolution and high accuracy for detection. It is anticipated that high field MRI contrast agents can achieve high performance in MRI imaging, where parameters of chemical composition, molecular structure and size of varied contrast agents show contrasted influence in each specific diagnostic test. This review firstly presents the recent advances of nanoparticle contrast agents for MRI. Moreover, multimodal molecular imaging with MRI for better monitoring is discussed during biological process. To fasten the process of developing better contrast agents, deep learning of artificial intelligent (AI) can be well-integrated into optimizing the crucial parameters of nanoparticle contrast agents and achieving high resolution MRI prior to the clinical applications. Finally, prospects and challenges are summarized.The ultra-high-field magnetic resonance imaging (MRI) nowadays has been receiving enormous attention in both biomaterial research and clinical diagnosis. MRI contrast agents are generally comprising of T As one of the most attractive and useful techniques for non-invasive imaging, magnetic resonance imaging (MRI) shows its great superiority in the practical application of clinic diagnosis, as well as the biomedical research  has advanced the exploration of ultra-high-field MRI, where AI in conjunction with MRI is supposed to be prevalently used in many cases, ranging from imaging reconstruction to the final clinical decision support Busch, . For ins2), longitudinal relaxivity (r1) and the ratio of r2/r1 are used to evaluate the contrast efficiency of MRI contrast agent, where a high ratio of r2/r1 generally results in a high contrast efficiency . The existence of electron spin angular momentum of adjacent ions contributes to an enlarged total electron angular momentum, leading to a high r1. On the contrary, total electron angular momentum will be quite small if contribution is only from electron orbital angular momentum.To reveal the efficacy of a variety of contrast agents, crucial parameters have been defined to indicate the efficiency and species of contrast agents. Transverse relaxivity  /Cy7.5-conjugated tobacco mosaic virus (TMV) was developed for ultra-high-field MRI, confirming its high transverse relaxivity r2 and suitable for both NIRF imaging and T2 MRI in cancel cells diagnosis  of contrast agent in water solutions at 7.0 and 9.4 T, showing a pronounced negative contrast gradient as a function of contrast agent concentration  reach to the maximum value for both T1 and T2 images at 60 min. Subsequently, the values rapidly drop at 240 min. This demonstrates the good metabolism of nanoparticle contrast agent in liver. Consequently, this kind of nanoparticles achieved through surface and interfacial engineering demonstrates its great promising as an ultra-high-field dual-modal MRI contrast agent for accurate diagnosis  and superior tissue penetration depth, while its sensitivity requires substantial improvement compared with the direct optical imaging technique. CT has the benefits of high resolution (30 ~ 400 μm) and 3D visual graphing; however, the low sensitivity also becomes the obstacle for its further wide application. Near-infrared fluorescence (NIRF) imaging share the benefit of high sensitivity, while it is constricted by the low spatial resolution. Computed tomography (CT) and MRI can provide the unparalleled structural information, while CT and positron emission tomography (PET) can offer provide the insight into exploring the morphological and functional behaviors. Moreover, other combinations of multimodalities have been also attempted to resolve different upcoming issues in clinics., such as PET/MRI system into the dynamics of brain  is proposed as a sensitive and non-invasive technique, which provides precise and specific identification of signals in combination with nanoparticle contrast agents. Iterative coating approach is used to reach the rational design and synthesis of core/shell magnetic nanoflower contrast agent   are reported to target both CD44 and folate receptor (FR)  , showingtor (FR) . MoreoveGiven the significance of β-glycosidases in human body, a set of β-glycosidase-specific activity-based probes (ABPs) are specifically studied to detect enzymatic activity over a range of glycosidases, demonstrating its role of tracking pathological relevant enzymes and its great potential in discovering the structural and biochemical functionality  is generally required to perform the scan with contrast agent, which will inevitably induce high cost of equipment purchase and high operational cost of long-tern scanning, and the unexpected unsafety issue owing to the high magnetic field. Until now, many attempts have been devoted to exploiting machine learning of AI to replace the high field scan process, where high resolution MRI can be obtained through AI deep learning of low-resolution MRI (Shen et al., 2+, Fe3+, Mn2+ based nanoparticles (Zhu et al., With the aim of developing contrast agents with good compatibility, biodegradability, high relaxivity for MRI and ultra-high-field MRI, continuous efforts have been devoted to exploring and searching for better contrast agents for MRI. To improve the imaging sensitivity of nanoparticle contrast agents, geometry of size, surface, shape of nanoparticles can be designed, and tuned via surface modification approaches, or other techniques (Zhou et al., In addition, based on the design principles of nanoparticle contrast agents, crucial aspects in terms of easy-processable and low cost are considered to meet the large-scale demands, which can be resolved with the assistance of AI technology. As the fabrications of nanoparticle contrast agents focus on optimizing a variety of parameters, with the emerging of AI, the algorithm based on the deep learning will complete all these tasks prior to perform the final optimized experiments for verification, which will save substantial time, efforts and achieve the high resolution MRI (Moser et al., Molecular imaging enables the quantitative characterization and measurement of biological diagnosis at the cellular and molecular level, which will inevitably advance the modern and future medical imaging and diagnosis (Wu and Shu, To sum it up, multimodal molecular imaging agents and specific targeted molecular imaging agent will be both expected in the molecular imaging in future clinic application. With the aid of AI, a major breakthrough can be expected in multimodal molecular imaging for molecular biology clinics, despite molecular imaging combined with AI learning is still at the initial stage.The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The nucleus of the solitary tract (NTS) is the primary brainstem centre for the integration of physiological information from the periphery transmitted via the vagus nerve. In turn, the NTS feeds into downstream circuits regulating physiological parameters. Astrocytes are glial cells which have key roles in maintaining CNS tissue homeostasis and regulating neuronal communication. Recently an increasing number of studies have implicated astrocytes in the regulation of synaptic transmission and physiology. This review aims to highlight evidence for a role for astrocytes in the functions of the NTS. Astrocytes maintain and modulate NTS synaptic transmission contributing to the control of diverse physiological systems namely cardiovascular, respiratory, glucoregulatory, and gastrointestinal. In addition, it appears these cells may have a role in central control of feeding behaviour. As such these cells are a key component of signal processing and physiological control by the NTS. The vagus nerve  innervates most of the major internal organs including the heart, lungs and gastrointestinal tract, and its afferent branch sends terminals to the NTS via a nerve bundle called the solitary tract (ST). The majority of the inputs from the vagus terminate in the caudal NTS while more rostral areas are innervated by the facial nerve  and glossopharyngeal nerve . Vagal afferents form glutamatergic synapses onto second-order neurons relaying information from the periphery to the NTS . NTS neu2+ coupled to gliotransmitter release [2+ variations have been demonstrated in response to neurotransmitters and lead to alterations in downstream signalling, including release of gliotransmitters  and modulation of glutamate transport [Astrocytes are glial cells that populate the entire CNS. These cells are crucial to brain function, providing structural and metabolic support to neurons and playing roles in synaptic transmission and cerebrovascular coupling  in second-order NTS neurons [Synaptic transmission between vagal afferent terminals and second-order viscerosensory neurons allows for the appropriate autonomic and behavioural response to physiological challenges. Vagal afferents entering the NTS via the ST release glutamate onto postsynaptic neurons. This can be modelled experimentally where in  neurons ,2. Astro2+ permeable AMPA receptors (AMPA-R). Using Ca2+ imaging from astrocytes and neurons in ex vivo NTS rat brain slices, McDougal and colleagues demonstrated that astrocytes can detect glutamate released following electrical ST stimulation and respond with increases in intracellular Ca2+, both from extra- and intracellular sources 2+ influx to the cell, which drives Ca2+-induced Ca2+-release further increasing Ca2+ levels via liberation from intracellular stores. These Ca2+ elevations are sensitive to the AMPA-R antagonist NBQX but not antagonists for metabotropic glutamate receptors (mGlu-R) 1 or 5 nor NMDA receptors (NMDA-R) NTS astrocytes express Ca2+ due to accumulation of citrate, a Ca2+ chelator 2+-signalling and gliotransmission in NTS astrocytes has been demonstrably and reversibly inhibited by 30 minute treatment of FC followed by a 10 minute washout period In addition to sensing ST-derived glutamate, astrocytes also contribute to transmission across the ST-NTS neuron synapse. Astrocyte function can be selectively inhibited with fluoroacetate (FAC) and its metabolite fluorocitrate (FC). FC inhibits the tricarboxylic acid (TCA) cycle and both FC and FAC preferentially affect astrocytes at nanomolar concentrations used in experimental settings A) A is inhibited when astrocytic glutamate uptake is reduced due to activation of extra-synaptic NMDA-Rs In the presence of FAC, both ST-evoked EPSC amplitude and spontaneous EPSC frequency are reduced in NTS neurons that project to the ventrolateral medulla (VLM) A second mechanism by which astrocytes may support ST-NTS neurotransmission is by active supply of the metabolite lactate threo-β-benzyloxyaspartic acid (TBOA) elevates synaptic glutamate as evidenced by NTS neuronal depolarisation and action potential firing, and increased spontaneous EPSCs 2 this may be a common feature of astrocytes that allows for chemodetection of low pH [A further mechanism by which astrocytes may modulate ST-NTS neurotransmission is by regulating synaptic glutamate availability. Astrocytes clear glutamate from the synaptic cleft and recycle it to neurons in the form of glutamine, which can then be used for further glutamate synthesis by neurons f low pH ,54.Thus, combined evidence from a number of independent research groups shows that astrocytes support synaptic transmission in the NTS by buffering and recycling synaptic glutamate 3Astrocytes show regional variations in the expression of the cytoskeletal glial fibrillary acidic protein (GFAP), which is dynamically regulated in response to local variations in the brain microenvironment Several studies have reported that NTS GFAP expression, commonly measured using immunoreactivity, is modulated by different experimental stimuli . For exaIn addition to hypoxic challenge, NTS GFAP expression is regulated in a diverse range of disease models associated with inflammation . For exaAstrocytes and their progenitors, radial glia, play a critical role in brain development In addition to those outlined above, a number of other studies have shown dynamic regulation of NTS astrocytes 44.12 allowing for compensatory homeostatic chemoreflex responses to deviations in O2 or CO2The NTS is the initial CNS detector of cardiovascular parameters . SpeAs discussed above, hypertension (in a rat model) influences expression of GFAP in the NTS In line with their role in integrating activity at the ST-NTS synapse, glutamate reuptake and recycling by NTS astrocytes is crucial for cardiovascular function. Blockade of all EAATs (by TBOA) or only EAAT2 (by DHK) causes cardiac depression and reduced baroreflex response to phenylephrine in rats A and greater amplitude NMDA and AMPA currents evoked by ST stimulation [A, reduces the amplitude of ST evoked AMPA currents and increases the amplitude of ST evoked NMDA currents. These effects were not observed in slices from rats maintained under sustained hypoxic conditions [Under hypoxic conditions, chemoreflex responses are engaged which initiate compensatory increases in respiratory and heart rate. This effect persists even when animals are returned to normoxia, suggesting some central adaptation to the low oxygen environment mulation ,40. Inhinditions ,40. ThisTogether these studies illustrate the necessity for tight control of synaptic glutamate in the NTS to maintain cardiorespiratory function, and the importance of astrocytes in this process. Furthermore, the published studies on adaptation to hypoxia suggest that astrocytes adapt to compensate for changes in physiological need.4.2Orchestrated in part by the brain, the counter-regulatory response to hypoglycaemia (CRR) is initiated when blood glucose falls below the normal euglycemic range and is a multifaceted hormonal and neuronal response to restore blood glucose. The hindbrain is a critical site of hypoglycaemia detection and is required to drive appropriate counter-regulatory responses (namely feeding and increases in blood glucose) ,86. In p2+ in response to low glucose or the non-metabolizable glucose analogue 2-deoxyglucose  indicating the low glucose-sensitivity of these cells 2+ increases in both cell types are diminished following treatment with FC in mouse brain slices th ventricle  increases blood glucose in anaesthetised rats, indicating that local reductions in brainstem glucose level are sufficient to drive compensatory changes in glucose homeostasis In rat brain slices, 40% of NTS astrocytes increase their intracellular CaTH). In brain slices from mice, the Ca2+ response of NTSTH neurons to 2-DG is abolished by pre-treatment with either FC or the broad spectrum P2 purinergic receptor antagonist suramin 2+ responses to low extracellular glucose and 2-DG in rat brain slices 2+ response to low extracellular glucose is downstream of protein lipase C, which indicates that GLUT2 may engage second messenger systems in addition to its transporter function TH neurons via purinergic gliotransmission in order to drive the appropriate CRR.Of critical importance to the CRR are NTS catecholaminergic neurons, identified by their expression the enzyme tyrosine hydroxylase  by AICAR. In addition, this effect is abolished by inhibition of AMPK with compound C A study using a mouse model expressing the fluorescent protein td-tomato in GLUT2-expressing cells found labelling of intrinsically glucose-inhibited GABAergic neurons in the NTS 2+ rises in low-glucose responsive astrocytes precede those of low-glucose responsive neurons by 50 seconds on average and a similar relationship is seen in NTS slices from mice [TH neurons (and other glucose sensitive NTS neurons e.g. GABAergic GLUT2 expressing neurons) to low glucose, at least in this experimental configuration [Thus, it appears that in concert with glucose-inhibited NTS neurons, astrocytes in the NTS are involved in sensing low glucose levels. In NTS slices from rats the intracellular Carom mice ,93. The guration ,93. In gguration ,46. This2+ signalling in astrocytes 2+ responses to extracellular low glucose or glucopivation are attenuated in glucose-inhibited NTS neurons and NTSTH neurons in brain slices from mice Influenced by Rogers and Hermann In vitro, human primary astrocytes show metabolic adaptations following recurrent exposure low glucose levels, which has been suggested reflect a compensatory response to preserve function While NTS astrocyte integration of glycaemic status appears important for the physiological response to low glucose, what remains to be determined is whether astrocyte glucose sensitivity is reduced after recurrent hypoglycaemia, which may lead to blunted CNS glucose sensitivity and attenuation of the CRR 4.32+ in 40% of NTS astrocytes in rat brain slices. Critically, NTS pre-treatment with FC abolished the inhibitory effect of exendin-4 directly delivered to the NTS on food intake. Taken together these data suggest that NTS astrocytes are a component of the central GLP-1 satiety system, although the molecular mechanism by which they exert their effects has not been fully investigated There is a large body of evidence implicating NTS neurons in the integration of viscerosensory signals from the stomach and gastrointestinal tract, including encoding of satiety and meal termination 2+ in response to application of ghrelin and leptin suggesting that, at least in vitro, brainstem astrocytes respond to a diverse range of hormonal signals that regulate appetite 2+ signal diversity in astrocytes is beginning to be understood and may provide insight into how ‘opposing’ stimuli are processed within a single cell [In addition to exendin-4, cultured rat brainstem astrocytes show increased intracellular Cagle cell ,110.et al and Cork et al reflect a species difference or are the result of two different methodological approaches to detect cells that express GLP-1R, but raises the possibility that astrocytes may play this important role in rats but not mice.Although fluoro-exendin-4 binds to astrocytes in the rat NTS via its G-protein coupled receptor ODN-GPCR Further support for a key role of NTS astrocytes in the control of feeding comes from a series of studies on endozepines. NTS astrocytes (and tanycyte like cells of the area postrema known as vagliocytes Work from our group indicates a role for NTS astrocytes in the control of food intake. We found that 12-hour consumption of a high-fat high-sucrose diet increases GFAP immunoreactivity and astrocyte process branching within the mouse NTS 2+ in neighbouring neurons via direct activation of NMDARs and by increasing presynaptic glutamate release In addition to integration of information on satiety and meal termination, the NTS also contributes to modulation of parasympathetic tone to the gastrointestinal tract via preganglionic motor neurons in the DMX (for review see 5The studies reviewed here clearly illustrate the importance of NTS astrocytes in both effective synaptic transmission  and physFurther work is needed to elucidate the mechanisms by which astrocytes in the NTS can alter their synaptic support functions in an activity-dependent manner; for example, do astrocytes regulate EAAT2 glutamate transport in response to AMPA-R activation? Also, of interest is the integration of neuronal and hormonal cues by NTS astrocytes since they have been shown to respond to both. The application of cell-type specific genetic tools for cellular monitoring and manipulation The authors of this manuscript declare no conflict of interest.


The ability of health systems to cope with coronavirus disease (COVID-19) cases is of major concern. In preparation, we used clinical pathway models to estimate healthcare requirements for COVID-19 patients in the context of broader public health measures in Australia. An age- and risk-stratified transmission model of COVID-19 demonstrated that an unmitigated epidemic would dramatically exceed the capacity of the health system of Australia over a prolonged period. Case isolation and contact quarantine alone are insufficient to constrain healthcare needs within feasible levels of expansion of health sector capacity. Overlaid social restrictions must be applied over the course of the epidemic to ensure systems do not become overwhelmed and essential health sector functions, including care of COVID-19 patients, can be maintained. Attention to the full pathway of clinical care is needed, along with ongoing strengthening of capacity. As of late September 2020, >30.6 million confirmed cases of coronavirus disease (COVID-19) were reported worldwide, involving all global regions and resulting in >950,000 deaths  services to inpatient units and ultimately ICUs. Stresses on clinical care provision will result in increased illness and death  of 2.53 and a doubling time of 6.4 days  paradigm . Transmi6.4 days . Potentiumptions . For eac<72 hours. We assumed that 80% of identified contacts adhered to quarantine measures and that the overall infectiousness of truly exposed and infected contacts was halved by quarantine, given delayed and imperfect contact tracing and the risk for transmission to household members.We simulated a case-targeted public health intervention. Cases were isolated at the point of diagnosis. We assumed isolation occurred 48 hours after symptom onset, limiting the effective infectious period and reducing infectiousness from the point of identification by 80%, enabling imperfect implementation. Targeted quarantine of close contacts was implemented in the model framework by dynamic assignment of a transient “contact” label. Each time a new infectious case appears in the model, a fixed number of temporary contacts are labeled. Only contacts can progress through the exposed and infectious states, however, most remain uninfected and return to their original noncontact status >60% of all infections requiring ICU admission occur in persons >70 years of age ; total ICU capacity expansion to 200% of baseline, tripling the number of beds available to treat COVID-19 patients (3× ICU capacity); or total ICU capacity expansion to 300% of baseline, increasing by 5-fold the number of beds available to treat COVID-19 patients (5× ICU capacity).We also considered a theoretical alternative clinical pathway, COVID-19 clinics, which had constraints on bed numbers but double the capacity to assess severe cases in hospitals. The purpose of including this pathway was to reveal unmet clinical needs arising when bed block constrains ED triage capacity, potentially preventing needed admissions to the ICU.Broad based social distancing measures overcome ongoing opportunities for transmission arising from imperfect ascertainment of all cases and contacts, and from presymptomatic and asymptomatic persons. In settings where nonpharmaceutical social interventions have been applied, associated case-targeted measures also have been in place, making the effectiveness of each difficult to quantify (Therefore, we focused on the overall objective of distancing, which is to reduce the reproduction number. We modeled the effect of constraining spread by 25% and 33%, overlaid on existing case-targeted interventions, which is consistent with observed impacts of combined measures less restrictive than total lockdown (According to our model, an unmitigated COVID-19 epidemic would dramatically exceed the capacity of the health system of Australia over a prolonged period . Case isIncreasing the number of ICU beds available to patients with COVID-19 reduces the time over which ICU capacity is anticipated to be exceeded, potentially by more than half . The dur>20%–40% if capacity increases by 5-fold (These figures do not accurately reflect the true requirement for services, however, because blocks in assessment pathways resulting from ED and ward overload are an upstream constraint on incident ICU admissions. The alternative triage scenario, the COVID-19 clinic, reveals a high level of unmet clinical need for both ward and critical care beds given baseline bed capacity , 4. Casey 5-fold  Figure 3y 5-fold  Figure 4Our simulated scenarios show that case isolation and contact quarantine alone will be insufficient to keep clinical requirements of COVID-19 cases within plausibly achievable expansion of health system capacity, even if very high and likely unrealistic levels of case finding can be maintained. We therefore explored the effects of additional social distancing measures that reduced input reproduction numbers by 25% and 33% on ICU requirements in relation to the same clinical care capacity constraints . SimulatThe overlay of distancing measures, applied from the initial stages of the epidemic and maintained throughout, suppresses epidemic growth to a level that is within the range of plausible ICU capacity expansion. The duration of ICU exceedance remains long in the 25% case , but thiThis modeling study shows that an unmitigated COVID-19 epidemic would rapidly overwhelm Australia’s health sector capacity. Case-targeted measures including isolation of those known to be infected, and quarantine of their close contacts, must remain an ongoing cornerstone of the public health response. These interventions effectively reduce transmission but are unlikely to be maintained throughout the epidemic course at the high coverage modeled here. As public health response capacity is exceeded, greater constraint of disease spread will be essential to ensure that feasible levels of expansion in available healthcare can maintain ongoing system functions, including care of COVID-19 patients. Broader based social and physical distancing measures reduce the number of potential contacts made by each case, minimizing public health workload and supporting sustainable case-targeted disease control efforts.Our findings are consistent with a recently published model (Much attention has been focused on expansion of available ICU beds per se, but our clinical model reveals that critical care admissions are further limited by the ability to adequately assess patients during times of system stress. In line with model recommendations, Australia, along with other countries, has implemented COVID-19 clinics as an initial assessment pathway to reduce impacts on primary care and ED services (Quantitative findings from our model are limited by ongoing uncertainties about the true disease pyramid for COVID-19 and a lack of nuanced information about determinants of severe disease, which we represented by age as a best proxy. The clinical pathways model assumes that half of available bed capacity is available for patients with the disease but does not anticipate the seasonal surge in influenza admissions that might be overlaid with the epidemic peak, although even in our most recent severe season, 2017, only 6% of hospital beds were occupied by influenza cases (Our model indicates that a combination of case-targeted and social measures will need to be applied over an extended period to reduce the rate of epidemic growth. In reality, the stringency of imposed controls, their public acceptability, and compliance, likely will all vary over time. In Australia, compliance with isolation and self-quarantining was largely on the basis of trust in the early response during February–March, but active monitoring and enforcement of these public health measures is now occurring in many jurisdictions. Hong Kong and Singapore initiated electronic monitoring technologies from the outset to track the location of persons and enforce compliance (The effectiveness of multiple distancing measures, including lockdown, has been demonstrated in Europe, but the contributions of individual measures cannot yet be reliably differentiated (All these strategies, which combine to flatten the curve, will buy time for further health system strengthening and sourcing of needed supplies. Protecting the health and wellbeing of healthcare workers will be essential to ensure ongoing service provision. ICU capacity will need to be increased several-fold in anticipation of the looming rise in cases.Multiple challenges must be overcome along the path to delivering safe and effective COVID-19 vaccines, and the timeframe for availability is highly uncertain (Additional information on design and formulas used for a coronavirus disease model to inform transmission reducing measures and health system preparedness, Australia.