Rheumatoid arthritis (RA) is an autoimmune disease of the joints characterized by synovial hyperplasia and chronic inflammation. Fibroblast-like synoviocytes (FLS) play a central role in RA initiation, progression, and perpetuation. Prior studies showed that sirtuin 1 (SIRT1), a deacetylase participating in a broad range of transcriptional and metabolic regulations, may impact cell proliferation and inflammatory responses. However, the role of SIRT1 in RA–FLS was unclear. Here, we explored the effects of SIRT1 on the aggressiveness and inflammatory responses of cultured RA-FLS. SIRT1 expression was significantly lower in synovial tissues and FLS from RA patients than from healthy controls. Overexpression of SIRT1 significantly inhibited RA-FLS proliferation, migration, and invasion. SIRT1 overexpression also significantly increased RA-FLS apoptosis and caspase-3 and -8 activity. Focusing on inflammatory phenotypes, we found SIRT1 significantly reduced RA-FLS secretion of TNF-α, IL-6, IL-8, and IL-1β. Mechanistic studies further revealed SIRT1 suppressed NF-κB pathway by reducing p65 protein expression, phosphorylation, and acetylation in RA-FLS. Our results suggest SIRT1 is a key regulator in RA pathogenesis by suppressing aggressive phenotypes and inflammatory response of FLS. Enhancing SIRT1 expression or function in FLS could be therapeutic beneficial for RA by inhibiting synovial hyperplasia and inflammation. Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial hyperplasia, inflammation and progressive destruction of the cartilage and bone, which ultimately lead to irreversible joint deformities and functional loss. The cause of RA is unclear, although many risk factors are recognized including genetics, environment, hormones, and lifestyle . RA is aFibroblast-like synoviocytes  are a special type of mesenchymal-derived cells lining the internal synovium. FLS displays many markers of fibroblasts, including CD90, type IV and V collagens, and vimentin. FLS also shows characteristics distinct from other fibroblasts, including secretion of lubricin that lubricates the synovium, and expression of unique surface markers such as CD55, VCAM-1, cadherin-11, integrins, and their receptors. Studies from the past two decades established FLS as a prominent cellular participants in RA. RA–FLS directly participates in synovial hyperplasia and the production of cytokines that perpetuate local inflammation. RA–FLS also contributes to modulation of immune cells and proteolytic destruction of extracellular matrix, cartilage, and bone. Targeting RA-FLS has been recognized as a novel therapeutic approach with potentially improved clinical outcomes and less impact on systemic immunity [Sirtuin 1 (SIRT1) is an NAD-dependent deacetylase engaging in a wide range of cellular functions such as transcription, cell cycle, DNA replication and repair, metabolism, apoptosis, and autophagy. SIRT1 is ubiquitously expressed and functions as a link between extracellular signals and transcriptional regulation of target genes. Several prior studies implicated a key role for SIRT1 in collagen-induced arthritis ,7 and osThe objective of the present study is to systematically characterize the role of SIRT1 in RA-FLS, in order to provide insight for novel disease mechanisms and potential therapeutic targets for RA.2-enriched atmosphere. Cells between passages three to six were used for experiments. Visual examination of cell morphology under light microscopy and fluorescence activated cell sorting analysis of cells stained with anti-CD11b antibody  confirmed that FLSs accounted for more than 95% of the cells (data not shown).Experiments involving human subjects were carried out in accordance with the declaration of Helsinki and were approved by the Medical Ethical Committee of Jiangsu University. Synovial tissues were obtained from 12 patients with RA  during synovectomy or joint replacement surgery at Northern Jiangsu People’s Hospital affiliated to Yangzhou University. Healthy synovial tissues were collected from six knee joint trauma patients  during surgery and used as negative controls (NC). Signed informed consent was obtained from all participants. Synovial tissue samples were prepared as described previously . FLS wasThe SIRT1-overexpression vector (pCDNA3.1-SIRT1) and the blank vector pCDNA3.1 were transfected into RA-FLS cells using Lipofectamine 2000  according to the manufacturer’s instructions. Cells were harvested at 48 h post-transfection for mRNA and protein expression measurements.GAPDH, forward 5′–AGC CAC ATC GCT CAG ACA-3′, reverse 5′–TCT CCT GGG AGG CAT AGA CC-3′. For each sample, RT-qPCR were performed in triplicates, and the relative mRNA level was normalized to GAPDH using the 2C−ΔΔt method [Total RNA was extracted using TRIzol reagent  according to the manufacturer’s instructions and reversely transcribed into cDNA using PrimeScript RT reagent kit . RT-qPCR was performed using the SYBR Premix Ex Taq kit (Takara Bio Inc.) on a 7500 fast real-time PCR System . The primers used for the RT-qPCR were as follows: SIRT1, forward 5′-TGG ACT CCA CGA CGT ACT-3′, reverse 5′-TCT CCT GGG AGG CAT AGA CC-3′; t method .GAPDH (ab8245)  overnight at 4°C, followed by incubation with peroxidase labeled anti-rabbit or anti-mouse secondary antibodies  at room temperature. The relative protein expression was detected using an enhanced chemiluminescent solution  and quantified by ImageJ using GAPDH as loading control.Total protein was extracted using RIPA lysis buffer  and concentration normalized using the BCA protein assay kit . Equal amount of protein lysates (30 μg each lane) was resolved by 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes . The membranes were blocked in 5% nonfat milk in Tris-Buffered Saline with Tween-20  and incubated with primary antibodies against NF-κB p65 (ab16502), phospho-NF-κB p65 (Ser536) (ab76302), acetyl-NF-κB p65 (Lys310) (ab19870), SIRT1 (ab110304), and 4 cells/well and received pCDNA3.1-SIRT1 or pCDNA3.1 transfection at 100 ng/well. At 24, 48, and 72 h post-transfection, 10 μl/well Cell Counting Kit-8  solution was added and incubated for 4 h at 37°C. The absorbance at 450 nm was measured on an ELx800 Absorbance Microplate Reader .RA-FLS was seeded on 96-well plates at a density of 2 × 10RA-FLS was detached as single-cell suspension 48 h post-transfection and fixed in 75% ethanol overnight at 4°C. Fixed cells were washed and stained with 25 mg/ml propidium iodide  in PBS containing 0.1% Triton and 10 mg/ml RNase  on ice for 30 min in the dark. Cell cycle was analyzed by flow cytometry on a FACSCalibur system .For apoptosis assay, fixed and washed cells were detected using an annexin V-FITC apoptosis detection kit (BD Bioscience Pharmingen) and apoptosis rate was measured as the combined percentage of cells that underwent early apoptosis (FITC+/PI-) and advanced apoptosis and necrosis (FITC+/PI+). Activity of caspase-3 and caspase-8 was measured using colorimetric protease assay kits .Cell migration was determined by wound healing assay. Briefly, transfected cells were seeded on six-well plates and cultured to confluence. A wound was created by manually scraping the cell monolayer with a sterile 200 μl pipette tip. Cells were washed twice with PBS to remove floating cells and incubated in medium containing 1% FBS for 24 h. The wounds were imaged at two time points (0 and 24 h) with a phase-contrast microscope  equipped with a Nikon DS-5M camera. Cell migration was calculated as percentage of healed distance relative to the initial wounds over 24 h.4 transfected cells in serum-free DMEM were added onto the upper chambers of 8-μm diameter Transwell inserts precoated with Matrigel , and 0.5 ml of DMEM with 10% FBS was added to the lower chamber. After 48 h incubation at 37°C, cells on the bottom chamber were fixed with 70% ethanol, stained with 0.1% Crystal Violet, and photographed under an inverted microscope. The numbers of cells in five random fields per well were counted and averaged as the invading cell number.Cell invasion was analyzed using Transwell invasion chambers . Briefly, 3 × 10Cytokine levels in the supernatant of RA-FLS were measured 48 h post-transfection using ELISA kits for human TNF-α, IL-6, IL-8, and IL-1β  per manufacturer’s instructions.t-test was used to evaluate the significance of difference between two groups. One-way analysis of variance followed by a post hoc Tukey’s test was used to test the significance of differences among three or more groups. P<0.05 was considered as statistically significant.Results from at least three independent experiments were presented as mean ± standard deviation (SD). Statistical analysis was performed using the SPSS 19 software package . Student’s To test potential association between SIRT1 and RA, we first measured the expression level of SIRT1 mRNA in the synovial tissues and FLS obtained from 6 negative control individuals and 12 RA patients by RT-qPCR. Compared with NC, SIRT1 mRNA was significantly down-regulated in RA synovial tissues and in RA-FLS , implicaIn order to systematically study the role of SIRT1 in RA-FLS, primary RA-FLS culture was established using cells isolated from RA patients. Transfection of the SIRT1 overexpression vector significantly increased SIRT1 expression in RA-FLS at mRNA and protein level by more than 2-fold compared with the control vector A,B. To eBecause proliferation assays only reflect viable cell numbers and do not distinguish the contribution from cell death, we then assessed the effects of SIRT1 on RA-FLS apoptosis. RA-FLS transfected with pCDNA3.1 or pCDNA3.1-SIRT1 was stained for apoptosis makers and analyzed by flow cytometry. SIRT1 overexpression increased the percentage of cells at both early apoptosis E, right.in vitro scratch wound healing assay revealed reduced migration in SIRT1-overexpressing RA-FLS as compared with empty-vector-transfected cells. The mean relative migration distance was decreased by nearly half in SIRT1-overexpressing RA-FLS compared with control cells , and acetyl-NF-κB p65 . In RA-FLS, SIRT1 transfection significantly reduced the expression of NF-κB p65, p-p65, and Ac-p65 compared with the control transfection E,F.in vitro models derived from RA patients, we uncovered that, in addition to the suppression of RA-FLS proliferation, migration and invasion, SIRT1 also substantially reduced the proinflammatory cytokine production and proinflammatory NF-κB signaling in RA-FLS. Therefore, SIRT1 is a promising dual-effect target for both synovial hyperplasia and chronic inflammation in RA.RA is a chronic autoimmune disorder characterized by synovial hyperplasia, chronic inflammation, and progressive destruction of joints. FLS is a major cellular component that maintains the synovial homeostasis, and in the setting of RA, drives the synovial hyperplasia and inflammation progression. Emerging evidence indicate that inhibition of RA-FLS effector molecules might be beneficial for RA therapy . The preSynovial hyperplasia is characterized by increased proliferation and decreased apoptosis of RA-FLS. SIRT1 is NAD-dependent protein deacetylase that links transcriptional regulation to a variety of metabolic signals, such as nutrient deprivation, DNA damage, and oxidative stress . One keyThe effect of SIRT1 on RA-FLS apoptosis has been elusive. Several prior studies showed that SIRT1 down-regulated proapoptosis protein CYR61  and protChronic synovial inflammation is a hallmark of RA. Activated RA-FLS contributes to synovial inflammation by secreting proinflammatory cytokines that serve as kindling for an immune response. These cytokines in turn recruit and activate leukocytes to the RA synovium, amplifying local inflammation and tissue damage. Cytokines not only are responsible for the inflamed joints but also have profound systemic effects. Therefore, cytokines represent novel therapeutic opportunities at both local and systemic levels. Several biologic agents targeting TNF-α and IL-1 are already licensed for RA treatment, and others showed promise in clinical trials . UnfortuNF-κB is constitutively activated in RA and maintains a proinflammatory, proliferative, and damaging phenotype of RA-FLS ,34,35. NTo summarize, SIRT1 suppressed RA-FLS proliferation, migration and invasion, induced apoptosis and reduced proinflammatory cytokine secretion from RA-FLS. We found these protective effects were partially due to SIRT1 inactivation of the NF-κB pathway. Our findings have therapeutic significance, because the SIRT1-mediated effects on RA-FLS involve a dual mechanism targeting both synovial hyperplasia and inflammation. Novel approaches aiming at augmenting the protective effect of SIRT1 may therefore be a promising option for FLS-targeted therapy in RA.


Fibroblast-like synoviocytes (FLS) are essential cellular components in inflammatory joint diseases such as osteoarthritis (OA) and rheumatoid arthritis (RA). Despite the growing use of FLS isolated from OA and RA patients, a detailed functional and parallel comparison of FLS from these two types of arthritis has not been performed.In the present study, FLS were isolated from surgically removed synovial tissues from twenty-two patients with OA and RA to evaluate their basic cellular functions.−CD31−CD146−CD235a−CD90+PDPN+). OA FLS and RA FLS at the same passage (P2-P4) exhibited uniform fibroblast morphology. OA FLS and RA FLS expressed a similar profile of cell surface antigens, including the fibroblast markers VCAM1 and ICAM1. RA FLS showed a more sensitive inflammatory status than OA FLS with regard to proliferation, migration, apoptosis, inflammatory gene expression and pro-inflammatory cytokine secretion. In addition, the responses of OA FLS and RA FLS to both the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-α) and the anti-inflammatory drug methotrexate (MTX) were also evaluated here.Pure populations of FLS were isolated by a sorting strategy based on stringent marker expression (CD45The parallel comparison of OA FLS and RA FLS lays a foundation in preparation for when FLS are considered a potential therapeutic anti-inflammatory target for OA and RA. Rheumatoid arthritis (RA) is a chronic autoimmune disease, and the inflammatory synovium is full of hyperplastic fibroblast-like synoviocytes (FLS) . MoreoveFLS stem from the mesenchymal lineage and exhibit aggressive and invasive cellular characteristics, secreting a range of cytokines and matrix factors that recruit other immune cells to the inflammatory synovium, eventually leading to cartilage injury and bone erosion –5. ThereThe aims of this study were to comprehensively characterize and compare the cellular function and inflammatory phenotype of FLS from the synovium of OA and RA patients by using a stringent sorting method. In particular, we were interested in investigating the proliferation, migration, apoptosis, and expression and production of inflammatory markers as well as in determining how this inflammatory phenotype is maintained over prolonged cell culture (P2-P4) and in response to pro-inflammatory cytokines and anti-inflammatory drugs.n = 23) include in this study were diagnosed with RA according to the 2010 American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) criteria [n = 23) conformed to the criteria from the American College of Rheumatology (ACR) [Synovial tissues were obtained from OA and RA patients  upon joint replacement or synovectomy at the First Affiliated Hospital of USTC and the First Affiliated Hospital of Anhui Medical University, Hefei, China. This study was approved by the Ethics Committee of the First Affiliated Hospital of USTC and the First Affiliated Hospital of Anhui Medical University. The informed consent was signed by each of the patients and their guardians. All RA patients (criteria . The diagy (ACR) , radiogr2. Culture media were replenished every 2 days, and cells were subcultured when they reached 90% confluence.The method used for the isolation of FLS from synovial tissues was modified from a method previously described . The syn8 total synovial cells from RA and OA synovium were used. It’s around (1.3 ± 0.4) × 108 and (1.0 ± 0.3) × 108 total count (events) in gate P4 for RA samples and OA samples. Isotype controls were included in the FACS detection for those proteins with relatively low expression -, phycoerythrin (PE)- or allophycocyanin (APC)-conjugated antibodies at 37 °C for 1 h: CD45-FITC (5 μg/test), CD31-FITC (5 μg/test), CD146-FITC (5 μg/test), CD235a-FITC (5 μg/test), PDPN-PE (2 μg/test), CD90-APC (2 μg/test), CD106 (VCAM1)-FITC (5 μg/test), CD54 (ICAM1)-PE (2 μg/test). All flow antibodies were from Miltenyi Biotechnology Company . After 3 times of washing by pre-cooled PBS, the cells were analysed by flow cytometer (BD FACSAria II) and BD FACSDiva software. For each test, 2 × 10OA and RA synovial tissues were fixed with acetone for 15 min, washed twice with PBS, and then incubated for 1 h in a humid chamber with primary antibodies . The synovial tissues were then washed 3 times with PBS and incubated for an additional hour with an isotype-matched horseradish peroxidase (HRP)-conjugated secondary antibody . After 3 additional washes, the HRP reaction was developed with diaminobenzidine (DAB) per the manufacturer’s instructions.Paraffin-embedded OA and RA synovial tissues were sliced and then blocked with 5% goat serum for 30 min. The tissues were incubated in primary anti-PCNA (Santa Cruz Biotec), anti-CD90 (Santa Cruz Biotec), anti-ICAM1  or anti-VCAM1  antibodies overnight at 4 °C. IHC staining was generally performed as previously reported. The primary antibody was omitted in negative controls. H&E staining and Masson staining were performed according to standard protocols.The proliferation abilities of OA and RA FLS were evaluated by CCK-8 assay  and Ki-67 staining (Miltenyi Biotec). All procedures were performed according to standard protocols.4 FLS were seeded into the upper chamber in serum-free medium, and serum-containing medium was added into the lower wells. After 24 h or 48 h, the upper chamber was washed with PBS twice and fixed with 4% paraformaldehyde for 20 min, and then 1% crystal violet was used to stain the invasive cells.Transwell (Costar) cell culture plates were used for migration and invasion assays. A total of 5 × 106 cells were collected and washed twice with ice-cold PBS. The cells were then stained using the Alexa Fluor®488 Annexin V/Dead Cell Apoptosis Kit with Alexa Fluor 488 annexin V and PI (Miltenyi Biotec) for flow cytometry according to the manufacturer’s guidelines. The untreated cells served as a negative control for double staining.Annexin V/propidium iodide (PI) staining was performed for the detection of apoptotic cells. After the desired treatment, 1 × 10OA and RA FLS were lysed in SDS buffer. The protein concentrations were determined via a BCA kit . Equal amounts of cell lysates were run on a gel, transferred onto a PVDF membrane , and probed with the following primary antibodies: interleukin-1 beta (IL-1β) , interleukin-6 (IL-6)  and GAPDH . GAPDH served as a loading control. After incubation with secondary antibodies, signals were visualized by enhanced chemiluminescence (GE system).5 cells were seeded in a 12-well plate for 48 h. Supernatants were collected to measure the amounts of secreted IL-6 and TNF-α. IL-6 and TNF-α were detected via a human IL-6 ELISA kit and a human TNF-α ELISA kit (Miltenyi Biotec), respectively.A total of 2 × 10Cultures of OA and RA FLS were grown to confluence in 6-well culture plates. Cells were treated for 24 h with TNF-α (20 ng/ml) or Methotrexate . Twelve hours after stimulation, whole-cell RNA was collected by TRIzol (Life Technologies), and it was analysed by qRT-PCR for gene expression. GAPDH was used as an endogenous control. A reverse transcription kit and qRT-PCR dye were obtained from Takara Biomedical Technology Company .P < 0.05, P < 0.01 and P < 0.0001, respectively (Student’s t-test).The error bars mean ± Standard Error of Mean (SEM) of six independent experiments. *, ** and *** indicate −CD31−CD146−CD235a−CD90+PDPN+ FLS [Twenty-three joint synovium biopsies from OA and RA patients were used in this experiment. H&E staining showed the typical pathological status of OA and RA hyperplastic synovial tissues Fig. a. MassonDPN+ FLS  after co+ cells was higher in RA synovial tissues than in an OA synovial tissues  resulted in a significant increase in the proliferation of both OA and RA FLS, and the inductive effect in RA FLS was greater Fig. a and b. −CD31−CD146−CD235a−CD90+PDPN+). OA FLS and RA FLS were compared and characterized based on multiple criteria to provide optimal confirmation of their origin and purity. The markers used in the FLS sorting strategy included general stromal fibroblast markers such as PDPN and CD90 [For inflammatory joint diseases such as OA and RA, FLS are an essential part of inflammation and joint erosion . Human Fand CD90  and moreand CD90 . The anaIn addition to surface markers of FLS, the present study also performed side-by-side comparisons of some basic cellular features of OA FLS and RA FLS, analysing proliferation, migration, expression/secretion of inflammatory cytokines, and response to pro-inflammatory cytokines and anti-inflammatory drugs. In general, RA FLS shows more aggressive cellular behaviour compared to OA FLS, including a more rapid proliferation rate, stronger invasive ability, and higher expression and secretion of inflammatory cytokines. These observations are consistent with our knowledge of the arthritis features exhibited by OA and RA. Moreover, higher expression of inflammatory markers, such as CCL2, IL-6, IL-1β and TNF-α, were also observed in RA FLS when compared to FLS isolated from the less inflamed OA synovium. OA FLS and RA FLS also show different responses to the pro-inflammatory cytokine TNF-α or the anti-inflammatory drug MTX. TNF-α could induce proliferation/migration of both OA FLS and RA FLS, while the inductive effect on proliferation was more obvious in RA FLS. In contrast, MTX could inhibit RA FLS proliferation but did not affect the proliferation of OA FLS in vitro. The different responses to MTX were further investigated with apoptosis experiments: MTX greatly induced apoptosis of RA FLS and induced less apoptosis in OA FLS. However, low concentrations of MTX (10 μM) only slightly promoted apoptosis of RA FLS and OA FLS (data not shown). The high dose of MTX can induce a series of severe side effects , which iMTX also inhibited up-regulated inflammatory markers . TNF-α sAlthough these inflammatory genes and markers have been previously validated with mice FLS culture in vitro , it is uIn summary, we characterized FLS isolated by collagenase digestion of synovial tissues from OA and RA patients by using a strict sorting strategy. The main cellular features were compared between OA FLS and RA FLS here. The current report provides an isolation and characterization standard for future research on human OA and RA FLS.By using a stringent sorting strategy, we comprehensively characterized and compared the cellular function and inflammatory phenotype  of FLS from the synovium of OA and RA patients in vitro. The parallel comparison of OA FLS and RA FLS lays a foundation for when FLS are considered potential therapies for anti-inflammatory treatment of OA and RA.Additional file 1: Figure S1. The isotype controls were included in the FACS detection for proteins with relatively low expression .


Optoacoustic imaging, based on the differences in optical contrast of blood hemoglobin and oxyhemoglobin, is uniquely suited for the detection of breast vasculature and tumor microvasculature with the inherent capability to differentiate hypoxic from the normally oxygenated tissue. We describe technological details of the clinical ultrasound (US) system with optoacoustic (OA) imaging capabilities developed specifically for diagnostic imaging of breast cancer. The combined OA/US system provides co-registered and fused images of breast morphology based upon gray scale US with the functional parameters of total hemoglobin and blood oxygen saturation in the tumor angiogenesis related microvasculature based upon OA images. The system component that enabled clinical utility of functional OA imaging is the hand-held probe that utilizes a linear array of ultrasonic transducers sensitive within an ultrawide-band of acoustic frequencies from 0.1 MHz to 12 MHz when loaded to the high-impedance input of the low-noise analog preamplifier. The fiberoptic light delivery system integrated into a dual modality probe through a patented design allowed acquisition of OA images while minimizing typical artefacts associated with pulsed laser illumination of skin and the probe components in the US detection path. We report technical advances of the OA/US imaging system that enabled its demonstrated clinical viability. The prototype system performance was validated in well-defined tissue phantoms. Then a commercial prototype system named Imagio™ was produced and tested in a multicenter clinical trial termed PIONEER. We present examples of clinical images which demonstrate that the spatio-temporal co-registration of functional and anatomical images permit radiological assessment of the vascular pattern around tumors, microvascular density of tumors as well as the relative values of the total hemoglobin [tHb] and blood oxygen saturation [sO2] in tumors relative to adjacent normal breast tissues. The co-registration technology enables increased accuracy of radiologist assessment of malignancy by confirming, upgrading and/or downgrading US categorization of breast tumors according to Breast Imaging Reporting And Data System (BI-RADS). Microscopic histologic examinations on the biopsied tissue of the imaged tumors served as a gold standard in verifying the functional and anatomic interpretations of the OA/US image feature analysis. The American Cancer Society (ACS) estimates that 40,290 women will die from breast cancer in the United States ,2. MammoDiagnostic US is widely performed in the workup of abnormal mammography findings. Advantages of US include its safety, convenience and capability to visualize tumors with video rate display that are radiologically occult, lack of ionizing radiation, and relatively low cost. Targeted diagnostic breast ultrasound helps in classifying breast cancer with excellent sensitivity, but suffers from low specificity. Ultrasound diagnosis of breast cancer has been primarily based on the lesion morphology (shape characteristics and ultrasound properties). Many malignant breast masses are too small to present sufficiently distinctive features on conventional ultrasound. Thus, the positive predictive value of the diagnostic ultrasound imaging after mammography and diagnostic ultrasound in biopsied masses (PPV3) is under 30% . When th1.1We present a newly developed technology of combined optoacoustic plus ultrasound (OA/US) imaging, integrated in the Imagio™ breast imaging system, specifically designed for imaging of breast and diagnosis of breast masses. This technology provides a two-fold enhancement of the overall diagnostic accuracy, combining specificity from functional imaging with molecular specificity of hemoglobin and oxyhemoglobin with the 95-plus% sensitivity of breast ultrasound anatomical imaging . We desc1.2For clinical radiologists, “seeing is believing”. Therefore, optical imaging technologies are naturally suited for medical applications. However, due to optical scattering, pure optical modalities that illuminate and sense light are not able to achieve adequate depth penetration and spatial resolution in the thickness of breast tissue necessary for complete evaluation of normal breast tissue . Optoaco1.3In the early years of development, OA imaging researchers realized that B-mode gray scale ultrasound imaging based on contrast provided by the acoustic impedance is complementary to the nature of medical information provided by the functional optoacoustic images ,16. The Furthermore, combining the two systems in one modality is acceptable to radiologists because they can readily adapt and associate functional information with morphology provided by co-registration of the optoacoustic and ultrasound images. With this understanding, a number of groups developed optoacoustic ultrasonic dual modality systems based on commercial ultrasound machines and commercial pulsed lasers . While 1.4in situ stage for years before switching into a rapid growth stage when the rate of the cell growth is much greater than the rate of apoptosis and requires new capillary blood vessels. Tumor-associated neovascularization allows the tumor cells to express their critical growth advantage. There is a constant requirement for vascular supply in cancerous tumors. Experimental and clinical evidence suggests that the process of metastasis is also angiogenesis-dependent ) may be used for detection of tissue abnormalities such as hypoxia and angiogenesis of cancer. Therefore, imaging of vasculature, blood circulation and blood distribution in tissues and measurements of [tHb] and [sO2] by methods of functional imaging, can be used for detection of microvascular network of aggressively growing malignancies in the breast and for their differentiation from normal tissues and benign tumors  and [Hb] molecules in the body. For purposes of functional biomedical diagnostics, parameters of the total hemoglobin, [tHb], and blood oxygen saturation, [sO2], can be determined from optoacoustic images acquired at multiple wavelengths, 2] can be measured from optoacoustic images acquired at two wavelengths, When spatial distribution of the optical fluence, c images . Since tll known , one can follows :(1)THb] and [tHb(r)] in the large tissue volumes including blood vessels and tumor microvasculature through normalization of the optical fluence distribution as a function of depth in the breast. We demonstrate below that functional imaging of tumor microvasculature and surrounding breast vasculature that is recruited to both feed and drain the tumor, together with depiction of [Hb] and [HbO2] concentrations, represents clinically relevant diagnostic information.In the past decade, there have been a number of optoacoustic studies in microscopy , endosco22.1The clinical system design was based on extensive computer modeling including Monte Carlo simulations of light propagation in the breast, signal generation, propagation and detection by realistic ultrasound transducers and optimization of image reconstruction algorithms for limited view tomography ,34, expeWe achieved clinical viability of OA + US technology for real-time functional anatomical mapping of the breast through (i) development of ultrawide-band ultrasonic transducer arrays and their proper loading to an ultra-low noise analog preamplifiers with high input impedance; (ii) understanding and elimination of optoacoustic image artefacts associated with hand-held probe design, which resulted in substantially increased image contrast, (iii) signal processing that inverted OA signal distortions by the detection system and (iv) image reconstruction and post-processing that computation of functional images and their accurate temporal-spatial co-registration with B-mode ultrasound images. To produce OA/US images, the Imagio system required uniquely designed subsystems: a handheld duplex OA/US probe, a laser system, OA/US reception signal processing, and OA/US image formation. Each of these subsystems is significantly different from conventional ultrasound to enable the most challenging requirement of simultaneously providing high spatial resolution (sensitivity to high ultrasonic frequencies) and high contrast of volumetric brightness of relatively large objects (sensitivity to low ultrasonic frequencies).2.2Perhaps the most unique part of the combined optoacoustic-ultrasonic imaging system, compared to conventional ultrasound, is the specialized hand-held probe. This is also one of the most vital and challenging part of the design. We integrated a fiberoptic light delivery system with a multi-element linear array ultrasound transducer in a form that is comfortable for the clinician and patient, compact, and light, while delivering the best possible imaging performance. The probe supports sufficient imaging penetration depth in the breast, while simultaneously minimizing image “clutter” artefacts associated with proximity of optical and acoustic components.2 , through analyzes of references [−1 at 757 nm and ∼3.5 – 4.5 cm−1 for 1064 nm. Optical absorption coefficient of cancerous breast tumors −1] was found to be statistically 2- to 3-fold higher (0.130 ± 0.060 at 757 nm) than in the surrounding breast tissue, but only, only slightly higher (0.154 ± 0.089) at 1064 nm. The average tissue optical properties result in an average effective attenuation of the optical flux in the near-infrared spectral range of approximately 3 times per cm.Back-projected optoacoustic images without image processing compensation would otherwise display gradually decreasing brightness of voxels as a function of depth. To quantify this attenuation, we determined bulk breast tissue optical absorption coefficient ferences  and expeAcoustic attenuation has been studied by Foster et al . Based oAnalysis of the optical and acoustic properties of the breast shows that breast tissue effectively attenuates both near-infrared light and ultrasound. Therefore, the first step in the optoacoustic image processing was accounting for OA signal attenuation.We developed a method of the OA image brightness normalization that resulted in the equal brightness of the well-characterized objects on clinical images of the breast, such as artery cross-sections. The method includes the following steps: (i) segment the brightest objects on the image: blood vessels and tumors, (ii) calculate average pixel brightness in each horizontal row of pixels excluding that of segmented objects, (iii) normalize each pixel brightness by dividing it to the average value at each depth.To test this method, it was applied to an optical and acoustically simulated dataset. 2.6Imagio produces images for each laser wavelength . First a reference region is selected. Then the two optoacoustic images are converted into the three functional maps: one map of the total hemoglobin, [tHb], proportional to the density of microvasculature in breast tissues, and 2 different maps of blood oxygen saturation, [sO2] relative to its average background level. The color palette for display of functional images is designed to reveal the functional information to the radiologist in its easy to interpret color map. The RGBA color maps are used for the [sO2] (left) and [tHb] (right) as displayed in As depicted in In addition to demonstrating relative degrees of oxygenation-deoxygenation, OA can demonstrate normal and abnormal anatomy of vessels that lie within a breast mass and its surrounding tissues. Interpreting physicians can assess the anatomy of these OA-demonstrated vessels in addition to the degree of relative oxygenation-deoxygenation in order to refine their overall evaluation for risk of malignancy.2.7The co-registered functional color OA maps are interleaved temporally and presented simultaneously in real-time along with the gray scale (B-mode) ultrasound. Co-registration of optoacoustic and ultrasound images is performed with the video frame rate of 10 OA/US images per second (5 fps for each of the two laser wavelengths). Since the creation of a single complete OA frame requires a pulse from each of the 2 OA wavelengths, complete OA frame rates are 5 fps rather than 10 fps. The two lasers (see section 2.2.1) operating independently were synchronized to emit a pair of pulses at the wavelengths of 757 nm and 1064 nm with a short delay of 5 ms between them. During such a short period of time almost no motion occurs in the course of hand-held probe scanning on the skin surface of the breast, even when the probe is moved slowly across the breast. Thus, when the duration separating data acquired from each wavelength is small, the multi-wavelength pixelwise operations for conversion of optoacoustic images into functional images do not produce motion artefact noise. There is a difference in frame update rates of gray scale ultrasound and OA. The ultrasound frame rate varies with depth of field and other technical settings, but is generally at least 3 to 4 times the update rate of the OA images. While the OA images are robust enough to tolerate slow sweeping of the transducer across the breast during scanning, movement of the transducer that is too fast can result in slight temporal and spatial miss-registration of the OA images with the underlying gray scale ultrasound images. Though a potential problem, in clinical trials, this was dealt with merely by adequate training in scan techniques, and was found not a clinically significant problem .The computer display shows separately an anatomical ultrasound image and two functional images of [sO2] and [tHb] superimposed with the anatomical image. All images are presented on screen simultaneously, but in clinical studies, the readers evaluated and interpreted the images in the following sequence: (1) B-mode ultrasound, a conventional image of breast morphology that gives the first diagnostic indications to the radiologists, then (2) functional image of [tHb] superimposed with ultrasound, and (3) functional image of [sO2] superimposed with ultrasound. The commercial version of the developed imaging system displays the two optoacoustic images , plus a “combined” image of [sO2] where only those pixels are colored that have colors on the [tHb] image. Thus, the total number of images on the display panel is six, when conventional ultrasound, OA Short (757 nm) and OA Long (1064 nm) are also included. However, for the purposes of Imagio technology presentation here we discuss only the main 3 images on the display that play the key role in the diagnostic imaging of breast cancer. In summary, the co-registering and temporal interleaving of OA and gray scale ultrasound images is creating a real time oxygenation-deoxygenation blood map fused with an underlying gray scale anatomic image.33.1The capability of Imagio™ to display functional values of [sO2] relative to the median background values of normal breast tissues was studied in a phantom made of PVCP with optical and acoustic properties replicating average breast tissue ,41 and ci.e. green color images were observed in the [sO2] range of ∼100% to ∼91%, no color is the [sO2] range from ∼90% to ∼85%, and red color images are in the [sO2] range below ∼85%.Blood oxygenation was changed by gradual oxygenation of fully deoxygenated blood in the air. The level of [sO2] in the tubes was monitored with a commercial oximeter. In this experiment, blood in the top vessel was changed from ∼100% [sO2] level to ∼91% level, while [sO2] in the other vessel was varied from ∼50% to 85%. This phantom experiment demonstrated that brightness of the optoacoustic images and colors of functional images are displayed correctly through the entire range of depths in the phantom. A gradual decrease of the image contrast was observed with increasing depth of the tubes from the imaging surface.The results obtained in the phantom were used for validation pertaining to the system readiness for clinical studies. Not only did the system accurately present colors representing the blood oxygen saturation in the major vessels, the image [sO2] color did not switch or disappear in the case of low hematocrit, however [tHb] signal-to-noise ratio became weaker against the background at the lowest hematocrit levels. In the clinical study of tumor angiogenesis, it has been found that 70-μm to 150-μm size vasculature can show significant deoxygenation in aggressive solid tumors ,23. This3.2During the device investigation, several different clinical studies were conducted on patients. Following the system validation in phantoms, a clinical feasibility study was performed on patients with breast masses suspected for cancer based on screening mammography followed by breast ultrasound. Patients with Breast Imaging Reporting and Data System (BI-RADS) scores of 3, 4 or 5 were recommended for dual-modality functional anatomical imaging comparing noninvasive diagnosis from the OA and US co-registered images of Imagio™ with the gold standard of core biopsy. Based on the clinical feasibility data of an initial 79 patients, the system was calibrated and its functional imaging algorithm was trained, which means that the thresholds were determined for display of colors in the area of image maximum brightness of the total hemoglobin [tHb] distribution and its oxygen saturation [sO2]. Following the feasibility study, a multicenter clinical trial guided by the Food and Drug Administration was performed on over 2000 patients to determine sensitivity and specificity of noninvasive diagnosis of breast masses suspected for malignancy utilizing the co-registered optoacoustic functional and ultrasound morphological images. The statistical analysis of this clinical trial was reported in . Here weThe functional parameters provide strong information suspicious of malignancy. Therefore, optoacoustic functional images increase confidence of radiologist interpretation compared to that based on conventional ultrasonic morphological images. Biopsy confirmed invasive breast carcinoma. The tumor does not have a well-developed internal angiogenesis, but both the partial marginal/boundary zone deoxygenated blush and the presence of multiple radiating vessel within the peripheral zone have very high positive predictive values for malignancy in the range of 90+%. Optoacoustic imaging provided meaningful information for the radiologist to accurately diagnose malignancy.4The main advantages of optoacoustic imaging are its capabilities of displaying the presence or absence of tumor neovessels and their anatomy as well as displaying functional blood distribution and oxygen saturation properties based on optical absorption differences of hemoglobin and oxyhemoglobin at different laser wavelengths . The devIn summary, we developed and described a method of qualitative functional imaging and demonstrated clinical utility of functional parameters such as [tHb] and [sO2] displayed within morphological tissues structures in the breast. Addition of these functional images to the co-registered breast ultrasound images may have potentially transformative impact on breast cancer care in terms of accuracy of diagnosis and differentiation of malignant lesions. Other advantages of Imagio as a clinical modality that require a special report include relative image contrast independence on the age of a patient as well as equal system performance in all racial and ethnical groups.5By combining optoacoustic (OA) and ultrasonic (US) imaging capability in one system (OA/US), the first clinical modality was developed for functional imaging within specific morphological structures of the breast and thereby satisfied a long-standing need for a more accurate diagnostic imaging of breast cancer. The advanced system design features that enabled contrast and accuracy of functional images co-registered with anatomical images are: (i) the hand-held duplex probe containing an 128 element linear array transducer of ultrawide-band that is protected from interference of both the direct illumination by the light diffusely scattered from tissue back to the probe and the light leaking from the optical fibers inside the probe housing; (ii) sensitive, low noise electronics; (iii) the dual wavelength laser rapidly emitting near-infrared pulses one after the other targeting hemoglobin and oxyhemoglobin; (iv) software with sophisticated signal processing and image reconstruction algorithms enabling real time rate co-registered optoacoustic and ultrasound imaging. Clinical viability of this new technology was demonstrated initially in a feasibility study and then in multicenter clinical trials involving over 2000 patients recruited in 16 leading university hospitals and private diagnostic centers of the United States. The examples of the clinical results presented in this report show that optoacoustic functional images has the potential to provide clinically valuable enhancement of the diagnostic specificity of conventional breast ultrasound by either upgrading morphologically benign masses to cancer (and thus saving lives) or downgrading morphologically suspicious lesions to definitely benign masses  or simply confirming and/or elevating radiologist confidence during interpretation of breast masses based on morphology. Further technical advances of the technology described here are envisioned in the direction of more quantitatively accurate full view three-dimensional optoacoustic tomography systems. These advances could help to enable automated screening in addition to improved diagnostics, especially valuable to replace sensitive but not specific detection by MRI for patients with familial history of breast cancer and simultaneously dense heterogeneous breast .Authors - technology developers  disclose employment or equity position at Seno Medical Instruments, however, no apparent conflict of interests, and authors - clinical collaborators , declare no conflicts of interest.


Many viruses initiate interaction with target cells by binding to cell surface glycosaminoglycans (GAGs). Heparan sulfate (HS) appears to be particularly important in fibroblasts, epithelial cells and endothelial cells, where it represents the dominant GAG. How GAGs influence viral infectivity in HS-poor target cells such as macrophages has not been clearly defined. Here, we show that mouse cytomegalovirus (MCMV) targets HS in susceptible fibroblasts and cultured salivary gland acinar cells (SGACs), but not in macrophage cell lines and primary bone marrow-derived macrophages, where chondroitin sulfate was the dominant virus-binding GAG. MCK-2, an MCMV-encoded GAG-binding chemokine that promotes infection of macrophages as part of a gH/gL/MCK-2 entry complex, was dispensable for MCMV attachment to the cell surface and for direct infection of SGACs. Thus, MCMV tropism for target cells is markedly influenced by differential GAG expression, suggesting that the specificity of anti-GAG peptides now under development as HCMV therapeutics may need to be broadened for effective application as anti-viral agents. Herpesviridae family and chronically infects ~60–80% of adults [Human cytomegalovirus (HCMV) is the largest member of the f adults . Infectif adults . HCMV isf adults . Accordif adults .HCMV vaccine strategies have focused mainly on blocking cell entry by the virus. A generally accepted entry mechanism involves the binding of HCMV glycoprotein gH/gL complexes to cellular receptors, which triggers a conformational change in the HCMV gB protein, thereby inducing fusion of the viral envelope with the target cell membrane . In HCMVAntibody, viral vector and subunit vaccines based on gB, the trimer and the pentamer are currently under development with a special interest in the prevention of congenital HCMV . HoweverA third potential target for development of vaccines or therapeutics involves cellular glycosaminoglycans (GAGs), which mediate binding of many viruses to target cells . In thisBALB/cJ mice were obtained from The Jackson Laboratory . All mice were maintained under specific pathogen-free housing conditions at an American Association for the Accreditation of Laboratory Animal Care-accredited animal facility at the National Institute of Allergy and Infectious Diseases (NIAID) and housed in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals under the protocol LMI-8E approved on 31 December 2015 and annually renewed by the Animal Care and Use Committee of NIAID.g for 5 min, and then washed twice with PBS. SGACs obtained from 6 mice were resuspended in 10 mL of bronchial epithelial basal medium  supplemented with bronchial epithelial growth medium bullet kit  and plated in 96-well plates previously coated with 150 μg/mL of Cultrex Basement Membrane Extract . Cells were washed with PBS and media was replaced at 24 and 48 h after isolation. Cells were maintained in culture for three additional days before being used in experiments.Mouse fibroblast cell lines NIH-3T3 and M2-10B4 and the mouse macrophage cell line RAW264.7 were purchased from ATCC . NIH-3T3 and RAW264.7 cells were maintained in DMEM-Glutamax  supplemented with 10% FBS. M2-10B4 cells were cultured in RPMI-Glutamax  supplemented with 10% FBS. Salivary gland- and lung-derived fibroblasts (SGFBs and LGFBs) were isolated from BALB/cJ mice as previously described . PrimaryGaussia luciferase and it was previously demonstrated that they establish productive infection in tissue culture and in mice [g for 3 h) of supernatants from 100% infected M2-10B4 cells, and then further purified by ultracentrifugation  through a 15% sucrose cushion in virus standard buffer . The pellet was resuspended in 0.5–1.0 mL of VSB, aliquoted and stored at −80 °C. Viral titers were determined by plaque assays in M2-10B4 cells as previously described [The viruses MCMV-3D (MCK-2 knock out) and MCMV-3DR (wild type) were generously provided by Dr. Martin Messerle . These viruses were engineered to express  in mice ,45. Viruescribed .2, 50 mM NaCl, and 0.01% BSA; ChABC buffer: 50 mM Tris-HCl pH 8.0, 60 mM sodium acetate, and 0.02% BSA). Subsequently, cells were washed once with PBS to remove the enzyme and infected with MCMV-3D or MCMV-3DR at the indicated moi in RPMI supplemented with 10% FBS. The luciferase activity in the supernatant was determined 18 hpi as explained above. Of note, we noticed that the ChABC buffer weakens the cell attachment to the plate, which may result in the loss of some cells during washes and final luciferase levels lower than in cells treated with the HepII buffer (See Cells seeded in a 96-well plate were infected at the indicated multiplicity of infection (moi) with MCMV-3D or MCMV-3DR in RPMI supplemented with 2% FBS. After a 2 h viral adsorption at 37 °C, the cells were washed once with PBS to remove unbound viral particles and then incubated in RPMI 10% FBS. The luciferase activity in 10 μL of supernatant was determined 18 h post infection (hpi) using the Biolux Gaussia Luciferase assay kit  and a Mithras LB 940 luminometer . The relative luminescence units (RLUs) obtained in supernatants from mock-infected cells were subtracted from all samples during the analysis. To study the role of different cellular GAGs in the infectivity of MCMV, where indicated, cells were incubated prior to infection with 1 U/mL of heparinase II (HepII) or/and chondroitinase ABC (ChABC)  for 30 min at 37 °C in the corresponding reaction buffer  was measured in a FlexStation 3 microplate reader .To evaluate the presence of soluble GAGs in the culture media of M2-10B4 cells, we analyzed the capacity of M2-10B4-conditioned supernatants to block the binding of B18, a GAG-binding protein encoded by vaccinia virus , to hepa5 cells were incubated on ice for 20 min with buffer alone or increasing concentrations  of B18-His in PBS-staining buffer . Beforehand, cellular Fc receptors were blocked using TruStain FcX antibody solution . After washing with PBS, cell-bound protein was detected with an anti-His mAb  and an anti-mouse Alexa Fluor 488 secondary antibody . In total, 20,000 events were collected in a BD LSR Fortessa analyzer , and the data were analyzed using FlowJo software .The binding of B18-His to the surface of M2-10B4, RAW264.7 and BMDM cells was analyzed by FACS. 3 × 10Tjp1, Aqp5, Aqp3 and Amy1—was calculated in mouse salivary glands and cultured SGACs by qPCR. For this, the total RNA from whole submaxillary salivary glands of BALB/cJ mice and from 5 day cultures of SGACs was isolated with Trizol  following the manufacturer’s instructions. Genomic DNA was removed using the Turbo DNA-free kit . cDNA was synthesized from 500 ng of RNA using the SensiFast cDNA synthesis kit . Another 500 ng of RNA from each sample were mock treated without reverse transcriptase. The Cq values for the amplification of each acinar marker and the reference gene Gapdh were obtained in a CFX96 Real-Time System  using the SensiFast SYBR kit  and the following primer pairs: Gapdh F (5′-aactttggcattgtggaagg-3′) and Gapdh R (5′-acacattgggggtaggaaca-3′); Amy1 F (5′-gaaaagatgtcaatgactggg-3′) and Amy1 R (5′-accatgttccttatttgacg-3′); Aqp3 F (5′-cttctttgatcagttcataggc-3′) and Aqp3 R (5′-gggttgttataagggtcaac-3′); Aqp5 F (5′-atcttgtggggatctacttc-3′) and Aqp5 R (5′-tagaagtagaggattgcagc-3′); Tjp1 F (5′-ctgatagaaaggtctaaaggc-3′) and Tjp1 R (5′-tgaaatgtcatctctttccg-3′). The relative expression for each marker was calculated as 2(Cqmarker −CqGAPDH).The relative expression of 4 common markers of salivary acinar cells—4–108) of MCMV-3D and MCMV-3DR were incubated with NIH-3T3 and BMDM cells for 30 min on ice to prevent virus internalization. Cells were washed three times with cold PBS to remove unbound virus and DNA was isolated using QIAamp Blood kit . Cell-retained viral iE1 copies were quantified by qPCR as explained above and represented relative to the Gapdh copies calculated by interpolation in a standard curve generated with a pcDNA3.1-Gapdh plasmid. Alternatively, MCMV-3D and MCMV-3DR were incubated at moi = 0.5 with M2-10B4, NIH-3T3 or RAW264.7 plated in 24-well plates. After a 30 min incubation on ice, cells were profusely washed with cold PBS and collected with a cell scraper in 100 µL of RPMI 2% FBS. Cells were lysed by three cycles of freeze and thaw and lysates were clarified by centrifugation. M2-10B4 cells in 96-well plates were infected with increasing volumes of the input stocks or 25 µL of the lysates and the luciferase activity in the supernatants was measured 18 hpi, as explained above. The RLUs upon infection with the lysates were represented relative to the RLUs obtained per µL of input used for the infection of M2-10B4.The capacity of MCMV-3D and MCMV-3DR to bind to the surface of fibroblasts and macrophages was evaluated by qPCR or by reinfection of M2-10B4 cells with cell lysates. After purification, viral stocks used in qPCR-based assays were resuspended in PBS and treated overnight with 625 U/mL of benzonase  at 4 °C to remove free DNA. Then, viral titers were determined by qPCR as the number of copies of the viral gene iE1/mL, interpolating in a standard curve generated with a pcDNA3.1-iE1 plasmid, the Cq values obtained with increasing volumes of the viral stocks as template and the primers iE1 2F (5′-catctcctgtcctgcaacct-3′) and iE1 2R (5′-cttgggctgctgttgattct-3′). Subsequently, increasing viral copies (10Gaussia luciferase reporter viruses MCMV-3D (MCK-2 knockout) and MCMV-3DR (MCK-2 wild type) [MCMV is known to infect both mouse fibroblasts and macrophages. Moreover, using a pSM3fr bacmid-derived MCMV system, it has been reported that MCMV infection of macrophages, but not fibroblasts, is promoted by MCK-2 . Here, wld type) , which aNext, we investigated whether MCK-2-dependent or -independent MCMV infectivity depended on cell surface GAGs. For this, we first tested the infectivity of both MCMV-3D and MCMV-3DR in fibroblasts and macrophages treated beforehand with the GAG lyases HepII and ChABC to deplete specific types of GAGs from the cell surface. HepII removes heparin and HS , and ChATo determine whether the presence of soluble GAGs in cultured M2-10B4 fibroblast supernatants might contribute to the effects of GAG lyases on MCMV infectivity, we tested the influence of GAG lyase-treated M2-10B4 cell-free supernatants on the binding of purified B18 protein to heparin D. B18 isThe effect of GAG lyase treatment on MCMV infectivity in macrophages, however, fundamentally differed from our observations in fibroblasts and were different for the macrophage cell line RAW264.7 compared with primary BMDM. For RAW264.7 cells, neither HepII nor ChABC pretreatment affected the infectivity of either MCMV-3D or MCMV-3DR viruses B. This cAqp5) and amylase-1 (Amy1)  B. A redu1 (Amy1) C, which Since the depletion of specific GAGs from the surface of fibroblasts, macrophages and SGACs altered MCMV infectivity in an MCK-2-independent manner, and since cellular GAGs are the first point of contact for the virus, we reasoned that MCK-2 may not be involved in MCMV cell tethering. To confirm this, we performed MCMV–target cell binding experiments with fibroblasts and macrophages using qPCR to quantify the viral copies of MCMV-3D and MCMV-3DR retained on the cell surface. As shown in In the present study, we have demonstrated that MCMV infection of diverse mouse target cell types is markedly and differentially affected by cell surface GAGs. In particular, infectivity in primary mouse salivary gland- and lung-derived fibroblasts was inhibited by HepII pretreatment but enhanced by ChABC pretreatment. For primary salivary gland acinar cells, infectivity was reduced by HepII pretreatment but was unaffected by ChABC pretreatment, whereas the converse was observed for infectivity in primary mouse BMDM pretreated with these two GAG lyases. Cultured cell lines revealed two additional GAG lyase susceptibility patterns for MCMV infection: neither GAG lyase affected infectivity in the cultured mouse macrophage cell line RAW264.7, whereas, as for primary fibroblasts, the two enzymes oppositely affected infectivity in the cultured fibroblast cell line M2-10B4 (HepII pretreatment decreased whereas ChABC pretreatment increased infectivity). These results imply that HS and CS GAGs have differential target cell type-specific effects on MCMV infectivity, with HS being most important in supporting infection in primary fibroblasts and acinar cells and CS being most important in supporting infection in primary macrophages.We have previously reported that the MCMV chemokine MCK-2, like host chemokines, can bind directly to GAGs , and othMCK-2-deficient MCMV reaches significantly lower viral titers specifically in the salivary gland when compared to an MCK-2-expressing virus ,20. HoweThe cell type-dependent GAG effects on MCMV infection reported here might be explained by differential availability of GAGs in different target cells. In fact, consistent with our results, CS is usually the most abundant type of GAG in macrophages  ,54,55. MAnti-HS peptides have been engineered to block HCMV and MCMV cell attachment . However


New technologies are being touted as solutions to many societal challenges not least of which are ageing and health. However, the rapid development of new technologies is proceeding with little input from older adults. This presentation highlights the perceptions and attitudes of three age cohorts related to the continuous technological advancement of products intended to support active and healthy aging. Participants were 30-39 (n= 639), 50-59 (n=703), 70-79 (n=779) years-old randomly sampled from the Swedish population registry. Results showed both similarities and difference across generations. For example, 24%-35% of older adults would like to use home monitoring devices  to support active and healthy aging, compared to 35%-56% of younger groups. More than 82% of all groups highlighted the importance of involving intended users in the development process. Results can be used to support the needs and desires of current older adults and future generations.


The current article provides a brief summary of biopsychosocial gender differences in alcohol use disorder (AUD), then reviews existing literature on gender differences in treatment access, retention, outcomes, and longer-term recovery. Among psychotherapies for AUD, there is support for the efficacy of providing female-specific treatment, and for female-only treatment settings but only when female-specific treatment is included. However, despite mandates from the National Institutes of Health to do so, there is little work thus far that directly compares genders on outcomes of specific psychotherapies or pharmacotherapies for AUD. Although existing research has mixed findings on sex and gender differences in overall outcomes, there are more consistent findings suggesting different mechanisms of behavior change among men and women in AUD treatment and long-term recovery. Thus, more work is needed that attends to gender and sex differences, including planning studies that are structured to examine not only gender-differentiated outcomes in treatment response, but equally important, differences in treatment access and attendance as well as differences in mechanisms of change in drinking behavior. Clinicaltrials.gov.Between 1994 and 2017, the National Institutes of Health (NIH) issued mandates that biomedical researchers include female participants in clinical research,Diagnostic and Statistical Manual of Mental Disorders (DSM-5) —with past-year rates of 10% among women and 18% among men, and respective lifetime rates of 23% and 36%.Most recent epidemiological results indicate a higher prevalence among men than women of AUD—defined by criteria of the fifth edition of the American Psychiatric Association’s Regarding the terminology used in this article—“sex,” “gender,” and “recovery”—the NIH definition of sex refers to biological differences between females and males in chromosomes, sex organs, and endogenous hormones, whereas gender refers to more socially based roles and behaviors that may vary by historical and cultural contexts.10Regarding recovery from AUD, there is currently no consensus in definition of this term. Historically, recovery has been associated with Alcoholics Anonymous as “ongoing cognitive, emotional, behavioral, and spiritual reconstruction of the sobered alcoholic”Lastly, the research reviewed in this paper uses diagnoses from DSM-IV and DSM-5. Whereas DSM-IV described two distinct disorders—alcohol abuse and alcohol dependence—DSM-5 combines these into a single alcohol use disorder (AUD) with mild, moderate, and severe subclassifications reflecting the number of symptoms met. The main criteria change from DSM-IV is that DSM-5 eliminates alcohol-related legal problems and adds alcohol craving as a criterion for AUD. Lastly, although the search did not exclude international research, the majority of findings reviewed are from studies conducted and/or funded in the United States.Alcohol is consistently shown to have more negative effects on women’s health than men’s, even at weight-adjusted lower levels of alcohol exposure, partly due to gender differences in pharmacokinetics of alcohol.18Stress plays an important role in the development and maintenance of AUD among both men and women.Sex hormones affect all body systems directly and indirectly, and for women there appears to be a reciprocal effect of alcohol on sex hormones.21Women with AUD report higher levels of co-occurring psychiatric conditions than do men with AUD. Co-occurrences of mental health conditions with AUD were examined using data from two waves (2001–2002 and 2004–2005) of the National Epidemiologic Survey on Alcohol and Related Conditions (NESARC).26Among individuals with AUD, women are more likely than men to experience alcohol cravings in response to daily negative emotion and stress.29There are high rates of trauma among women receiving treatment for any substance use, and an estimated 25% to 55% of women in substance use treatment have PTSD.Research has found gender differences in the relationship between social networks, social support, and alcohol use. For example, compared to men, women with AUD are more likely to have a family history of AUD and a spouse with a history of AUD.Research has illuminated gender differences in the biopsychosocial factors contributing to the development of, and recovery from, AUD. The physical effects of alcohol are more pervasive for women than men, and sex-specific factors, such as sex hormones, have been associated with alcohol use. In terms of psychosocial differences, stress, trauma, and negative affect are particularly relevant contributors to alcohol use and development of AUD among women. Relatedly, there are gender differences in terms of rates of co-occurring mental health conditions, the rates of major depressive disorder among women with alcohol abuse being particularly high. These differences provide a context for understanding potential gender differences in AUD treatment and recovery and can be used to guide future research.A small percentage of individuals with AUD ever receive treatment, with past-year estimates of 7% of men and 5% of women with AUD receiving treatment40Among individuals who do enter AUD treatment, there are gender differences in clinical presentation. Women tend to have more severe alcohol and drug use histories, lower education and income, higher unemployment and housing needs, more children living at home, and higher parental stress, and they tend to be younger in age.44Data on gender differences in treatment retention are mixed, and most studies have been completed among samples with substance use disorder (SUD), meaning the results are not specific to AUD. For example, a review by Greenfield and colleagues reported no overall gender differences in SUD treatment retention but hypothesized that there would be different predictors and mediators of retention among men and women.45ClinicalTrials.gov for clinical trials on these AUD treatments, and reviewed publications from large clinical trials for AUD, to determine whether gender differences were analyzed and reported. Lastly, the authors searched for and reviewed reports of clinical trials, literature reviews, or meta-analyses on specific treatments to identify commentary or results regarding sex or gender. This was done to address the fact that analyses not yielding any significant gender differences may not have been identified using the search terms. Thus, for some treatments the authors were able to comment on null gender difference findings. Despite the NIH mandate to include females in biomedical research,The following review on outcomes of psychosocial treatments for AUD focuses on empirically supported treatments identified by American Psychological Association Division 12.Motivational enhancement therapy (MET) is a psychotherapy that helps patients resolve their ambivalence about engaging in treatment and reducing or stopping their substance use. Cognitive behavioral therapy (CBT) is an approach that focuses on the reciprocal effects of cognitions, emotions, and behaviors that maintain problem drinking. In treating SUD, CBT also focuses on identifying and resolving factors that reinforce or punish the substance use behavior and teaching both general coping skills and coping skills to negotiate drinking triggers. Twelve-step facilitation (TSF) treatment for AUD is based on the traditional Alcoholics Anonymous (AA) 12-step model and focuses on AA attendance, personalized spirituality, and guided introspection (“step work”).MET and CBT are among the most widely researched treatments for AUD;49Witkiewitz, Hartzler, and Donovan tested whether matching patients’ motivation level to CBT or MET was associated with better outcomes in the aftercare arm of Project MATCH.51A meta-analysis on controlled trials of brief motivational interventions examined gender as a moderator of treatment effect.54Couples-based approaches to the treatment of AUD are based in the assumptions that partners engage in malleable behaviors that reinforce and/or punish the client’s drinking behaviors, and that enhancing intimate relationships can improve problem-solving, enhance relationship functioning, and reduce likelihood of relapse. Behavioral couples therapy (BCT) and Alcohol BCT (ABCT) have been shown to be effective at increasing rates of abstinence from alcohol, decreasing alcohol-related problems, and improving relationship functioning.Several studies have tested ABCT separately among samples of men and women. An early study among men with alcohol dependence and their female partners compared three conditions: (1) ABCT, in which the spouse attended all sessions that included both alcohol- and marital-focused treatment; (2) full spousal attendance but alcohol-focused treatment only; and (3) minimal spousal involvement in alcohol-focused individual treatment.59ABCT also has been tested among women with AUD, and one study compared ABCT to a treatment arm in which women received individual CBT for AUD.Three medications are currently approved by the U.S. Food and Drug Administration for the treatment of AUD: acamprosate, naltrexone, and disulfiram. There are important gender differences in their bioavailability, distribution, metabolism, elimination,A meta-analytic study examined acamprosate for AUD treatment separately for men and women from a total of 22 studies,One of the first studies on naltrexone for AUD was a multicenter, placebo-controlled RCT of injectable naltrexone,69A third study tested high-dose naltrexone in men and women with co-occurring cocaine use disorder and AUD in a double-blind placebo RCT.72Thus, early studies suggested naltrexone for AUD was not as effective for women as for men, or that women may experience worse side effects, contributing to worse outcomes. However, more recent research has suggested that these effects may be due to study characteristics such as sample size or outcomes assessed. Baros, Latham, and Anton used data from two RCTs comparing a naltrexone plus CBT group and a placebo plus CBT group and found effect sizes favoring naltrexone in men compared to women on some outcomes (drinks per drinking day), but not others .A secondary analysis of COMBINE data tested treatment effects separately in men and women and found that both genders had better treatment response when they received naltrexone with either medication management or combined behavioral intervention , in comparison to placebo and any other combination of treatments.In 2016, Agabio et al. cited the low number of women in clinical trials on disulfiram that preclude evaluation of sex differences in efficacy and safety.Emerging digital and mobile models of treatment delivery include platforms such as telehealth sessions via videoconference; direct access computer programs such as CBT4CBT;78The preliminary research on access and use of AUD treatment via digital and mobile technologies suggests gender differences. For instance, a survey of members of an online social network site for women trying to resolve alcohol problems revealed that 47% of the site’s members had never tried any other form of support related to their drinking.Existing research suggests no major gender differences in terms of overall outcome in psychosocial or pharmacological treatments for AUD. However, this finding is qualified by the small number of studies that directly test gender differences and the low enrollment of women in clinical trials. Additionally, as demonstrated by secondary analysis of Project MATCH, moderating factors such as AUD severity and motivation may be differentially associated with outcomes for men and women.Recovery is a complicated construct, ill-defined and historically confined to a mutual care, 12-step “disease model” system that considers abstinence as the only viable outcome.Alcoholics Anonymous, the largest and most popular mutual help organization available, offers primarily mixed-gender meetings, but also some single-gender meeting options . However, AA meeting content is consistent across groups and does not necessarily include gender-specific content.Outcomes of single-gender versus mixed-gender AA meeting attendance have not been studied; however, studies on gender differences in treatment outcomes among attendees of mixed-gender AA have shown some significant results, including different moderators of attendance for men and women. One longitudinal study followed 466 men and women for 16 years who were initially untreated for problem drinking.Witbrodt and Delucchi followed participation in AA for 7 years and found that men were more likely to stop attending over the 7-year period.83In sum, research on gender differences in outcomes of AA attendance are mixed, but the most consistent findings suggest women are more likely to stay in AA longer than men, and there may be different moderators  of the efficacy of AA for men and women.In line with contemporary notions of AUD and SUD as chronic, relapsing diseases requiring a continuum of care, McKay and colleagues developed and tested stepped and continuing care interventions with various levels of intervention, including telephone counseling.In a sample of participants who used cocaine, most of whom were also alcohol dependent, McKay and colleagues found that women but not men benefited from telephone continuing care.Sliedrecht and colleagues conducted a review of 321 articles, published between 2000 and 2019, to examine the evidence for precipitants of relapse in AUD.91In another review, Walitzer and Dearing indicated that rates of alcohol relapse did not differ among men and women, but evidence did indicate different predictors of relapse by gender.92Gender differences in empirical studies on viability of non-abstinent forms of recovery have recently been studied. Analysis of gender differences in such studies needs to attend to different thresholds for risky or heavy drinking for men and women.93In a study of three clinical trials for AUD—including data from Project MATCH, the COMBINE study, and the United Kingdom Alcohol Treatment Trial—several baseline variables were tested as predictors of low-risk drinking; gender was not found to be predictive.95One study examined men and women with AUD between ages 55 and 77 in a private outpatient program.Issues such as co-occurring mental health conditions, social environment, sleep, and physical health are directly affected by problem drinking and are important independent outcomes reflecting quality of life (QoL). Literature reviews have shown that heavy drinking is associated with reduced QoL, which improves with reductions in drinking.Attention to gender differences among various forms of recovery (both in the 12-step model and in the treatment outcome literature)—including examination of abstinence, reduction of drinking, and/or secondary outcomes—has yielded some interesting results, but research is sparse so far. Predictors of relapse appear to differ between men and women, with women being more likely to relapse in response to interpersonal conflict and negative affect whereas men are more likely to relapse in response to isolation and both positive and negative affect. Also, although being married is a protective factor for men, it can act as a risk factor of relapse for women. Having at least one close friend to discuss drinking with is differentially helpful for women. Also, gender differences in treatment outcome and maintenance may depend on the outcome of interest (drinking or secondary outcomes) and the “form of recovery” studied.There are several behavioral treatments now known to be efficacious for AUD, but there is almost no examination of gender differences in the AUD psychotherapy process and mechanisms of behavior change in this research literature. For example, the authors of this paper found 49 articles published between 2000 and 2012 (26 published since 2010) studying mechanisms of change in CBT, Motivational Interviewing, or MET or examining general therapeutic alliance as a mechanism of change. Of these 49 articles, 22 were review or non-empirical papers and did not mention gender. Of the 27 empirical studies, seven (26%) provided no sample breakdown by gender, one study (4%) had an all-female sample, and 17 (63%) had mixed-gender samples . Furthermore, of these 17 mixed-gender studies, only five (29%) mentioned gender at all, typically as a statistical covariate. Since 2012, researchers have continued to examine mechanisms of change but generally have continued to ignore gender or used single-gender samples.The Women’s Recovery Group (WRG), a treatment for women with SUD (including AUD), examined mechanisms of change between men and women. WRG was compared to a traditional mixed-gender Group Drug Counseling (GDC) treatment in Stage I102Litt et al. studied Network Support Treatment (NST) for AUD, which is designed to help patients build social support networks for sobriety.Recent studies have investigated potential mechanisms of behavior change among female-only samples receiving CBT for AUD A recent review conducted by the RAND National Defense Research Institute examined 24 AUD RCTs to examine gender differences in outcome and found mixed results, with little evidence for systematic gender differences in treatment effects across studies.Our review and those by Greenfield and colleaguesAs suggested by Moyer and colleagues,It is also important to note that even among the studies that examined sex and gender differences, the sample sizes of women were often small, and analyses were likely underpowered. Given the historical differences in prevalence of AUD among men and women, this may have been justifiable in the past. However, the convergence of prevalence rates for lifetime AUD among men and women no longer justifies such small samples of women in treatment. Although studies may recruit men and women, women often comprised less than 50% of the sample, which makes it difficult to examine gender differences. If gender is considered a moderating factor, there must be enough men and women to statistically power the examination of interaction effects. Thus, in conducting clinical trials it may be important to enroll comparable numbers of men and women, with sufficient power to properly examine gender differences.Another consideration is single-gender treatment options, with female-only treatment most often a focus of research. This area of research has examined the delivery of treatment in a women-only setting, with or without including female-specific content  should be reported in AUD treatment outcome research.Research suggests gender differences in relapse precipitants. Furthering our understanding of biological, social, and psychological determinants of relapse based on gender has implications for personalized or tailored relapse prevention approaches.Clinical trials are mandated to recruit men and women, as well as analyze and report gender differences; however, the field needs to adhere more stringently to these mandates in future research. This involves consistent changes to methods such as intentional oversampling of women, randomization based on gender, and gender-specific analyses.The research reviewed here provides ample reason to believe that men and women recover from AUD differently. It is important to test and report gender differences when studying mechanisms of change—mediators, moderators, and active therapeutic ingredients—in AUD treatments.


Temperate phages engage in long-term associations with their hosts that may lead to mutually beneficial interactions, of which the full extent is presently unknown. Here, we describe an environmentally relevant model system with a single host, a species of the Roseobacter clade of marine bacteria, and two genetically similar phages (ɸ-A and ɸ-D). Superinfection of a ɸ-D lysogenized strain (CB-D) with ɸ-A particles resulted in a lytic infection, prophage induction, and conversion of a subset of the host population, leading to isolation of a newly ɸ-A lysogenized strain (CB-A). Phenotypic differences, predicted to result from divergent lysogenic-lytic switch mechanisms, are evident between these lysogens, with CB-A displaying a higher incidence of spontaneous induction. Doubling times of CB-D and CB-A in liquid culture are 75 and 100 min, respectively. As cell cultures enter stationary phase, CB-A viable counts are half of CB-D. Consistent with prior evidence that cell lysis enhances biofilm formation, CB-A produces twice as much biofilm biomass as CB-D. As strains are susceptible to infection by the opposing phage type, co-culture competitions were performed to test fitness effects. When grown planktonically, CB-A outcompeted CB-D three to one. Yet, during biofilm growth, CB-D outcompeted CB-A three to one. These results suggest that genetically similar phages can have divergent influence on the competitiveness of their shared hosts in distinct environmental niches, possibly due to a complex form of phage-mediated allelopathy. These findings have implications for enhanced understanding of the eco-evolutionary dynamics of host-phage interactions that are pervasive in all ecosystems. Temperate phages may engage in long-term association with their bacterial hosts that can lead to mutually beneficial interactions. It is well established that prophages can offer their hosts benefits, including resistance to superinfection by homologous phages –4 and enProphages have frequently been referred to as “time bombs” e.g., , in whicLysogeny is hypothesized to be prevalent in marine environments , 16, wheSulfitobacter sp. strain CB2047 and its infecting temperate phage ɸ-A were originally isolated from a phytoplankton bloom ) were monitored at two discrete time points preceding and following measurable culture lysis   at the same time point  Fig. . The ɸ-Aint Fig. . Similarint Fig. . CollectattB), within the 3′ end of a host tRNA-Leu gene   Table . In addiattB site. The ɸ-D prophage was not present, indicative of a substitution . In contrast, CB-A formed more robust biofilms relative to CB-D  of 5.13 µm, compared with 3.48 µm for CB-D. The maximum biomass thickness was also larger for CB-A than CB-D and had a larger range   Fig. .Fig. 4Ph6 (±2.08 × 105) PFU/ml and 5.2 × 105 (±8.5 × 104) PFU/ml respectively. CB-D cultures of the same growth states do not yield quantifiable phage using plaque assays, indicating values below the 45 PFU/ml limit of detection for the assay.Given the discrepancy in growth dynamics of the two strains, we next determined whether there were quantifiable differences in free-phage titers in CB-A and CB-D cultures. CB-A stationary and mid-log phase cultures yielded 1.63 × 109 (±2.18 × 109) copies/ml and 1.18 × 109 (±0.27 × 108) copies/ml, respectively. Head-to-head competition assays during growth on a surface showed an opposite response: co-culture biofilms had 29% and 55% greater biomass than CB-A and CB-D monoculture biofilms, respectively  in broth culture, the ratio of CB-A to CB-D gene copies were 3.26 (range 2.00–4.73) after 24 h of co-culture. The co-cultures were ~90% and ~65% lower than typical densities for monocultures of CB-A and CB-D, respectively Fig. . The numely Fig. . In addiely Fig. ; the bioE. huxleyi bloom from which CB-D and ɸ-A were originally isolated, samples were collected immediately following collapse of the phytoplankton bloom for genetic characterization of viral particles. Of the eight size-selected viral DNA fractions sequenced, reads from two libraries (~35 and ~75 kb size fractions) mapped to ɸ-A and ɸ-D genomes. From the ~35 kb library , 580 individual reads mapped to ɸ-A and 705 mapped to ɸ-D  that mapped to ɸ-A and ɸ-D, respectively, are likely the result of incomplete separation of DNA molecules during PFGE. These data indicate that viral particles from both phage types were present, at non-equal abundances, in natural populations.From the same induced ɸ-D Fig. . Of thesɸ-D Fig. . The smaViral-mediated lysis of microbial cells in marine systems leads to quantitatively important impacts on food webs and biogeochemical cycles . Yet, ouLysogenized bacteria are typically resistant to superinfection, that is secondary infection, by homologous phages [reviewed in pepG) encoded by ɸ-A is upregulated during superinfection relative to non-superinfected CB-D controls. Similarly, upregulation of the CB-D host genes recA and lexA indicates activation of the global SOS response, which has been shown to mediate the lysogenic to lytic switch in various bacteria [e.g., Salmonella enterica, Escherichia coli, and Pseudomonas aeruginosa; reviewed in xre-like), is below the limit of detection, perhaps suggesting a role for this gene’s product in suppression of phage lytic genes during the lysogenic state. As the ɸ-A and ɸ-D encoded transcriptional regulators lack conserved catalytic domains common to well characterized phage repressors, these proteins may be valuable targets for future studies aimed at deciphering the lysogenic-lytic switch in these Sulfitobacter-phage pairs. An aspect of lytic activation of prophages in response to superinfection that has not been explored in our, or other systems, is whether lytic infection and prophage induction occur simultaneously in an individual cell or within distinct subpopulations of cells, one undergoing lytic infection by an exogenous phage and the other undergoing lytic activation of a previously quiescent prophage. It is possible that a subpopulation superinfected with one phage could communicate with non-superinfected counterparts, thus initiating a lytic induction, a phenomenon that has only recently been reported for a Bacillus phage [Infection of CB-D with ɸ-A leads to the simultaneous production of ɸ-A and ɸ-D, indicative of both lytic infection and prophage induction. While we do not yet know the proteins that mediate the lysogenic-lytic switch in this system, gene expression assays from infected cell populations support quantitative measurements of phage abundance. A putative peptidase (us phage .Sulfitobacter sp. CB-D cultures) lack a prophage at the attB site (data not shown). Such individual cells would have increased susceptibility to lysogeny by ɸ-A invasion. Finally, integration of both prophages in tandem could result in the establishment of transient polylysogens. Due to an intrinsic instability arising from the high degree of nucleotide identity between the two phages, such presumed polylysogenic events might be expected to readily revert to a single phage type. Regardless of the apparent replacement mechanism, the relatively high frequency with which new lysogens are recovered from superinfections suggests either genotypic switching is prevalent with this two-phage-one-host system or that one-host-phage pair displays higher fitness than the other in a given environmental context.In addition to a mixed infection resulting in the production of both ɸ-A and ɸ-D viral particles, infection of CB-D with ɸ-A also yields new lysogens in which the prophage appears to have been replaced by the superinfecting phage. The mechanism whereby this presumptive substitution occurs is not yet clear, but several possibilities exist. It could have been achieved through homologous recombination between the phage genomes, an oft-cited mechanism of viral evolution –63. AlteOur data indicate a competitive interaction between the two host-phage pairs based on a fundamental difference in their lysogenic-lytic switches. One manifestation of these differences is altered frequencies of SPI that influence growth dynamics when the strains are cultivated planktonically and as biofilms. Rates of SPI are anticipated to be the combined result of stochasticity in gene expression (genetic noise) and induction of the SOS response. It has been observed that either a drop in phage repressor protein levels below a given threshold concentration or sporadic expression of integrase genes may initiate the lytic cycle . Noise iS. enterica, which is important for the evolution and diversity of host populations [S. enterica studies demonstrate selective eradication of nonimmune hosts in mixed populations as a means of a competing strategy by Gifsy 2 lysogenized strains [P. aeruginosa strains lysogenized by Liverpool Epidemic Strain prophages use the phages as anti-competitor weapons against phage-susceptible P. aeruginosa populations in a chronic lung infection model [E. coli MG1655 lysogenized with λ reveal the competitive nature of this type of inaction is anticipated to be limited, as lysogenization of susceptible hosts ultimately diminishes nonlysogenized “competitors” [Hydra vulgaris, one microbiome member, a Curvibacter species, possesses an inducible prophage that lytically infects another microbiome member, a Duganella strain. Mathematical modeling predicts this interaction may modulate competition amongst microbiome members [Until recently, SPI was largely considered detrimental as some fraction of the cells is continuously lost by phage-induced lysis. However, benefits of SPI on bacterial fitness are now recognized, and include the release of extracellular DNA, which facilitates and enhances biofilm formation , 66, conulations . Relevanulations . As an eulations , 69. Emp strains . Rat modon model . Studiesetitors” . Finally members .Sulfitobacters, in particular, thrive in nature [Our system proposes a new element to this type of interaction: the reciprocal attack by genetically similar phages that share an integration site in a common host. Head-to-head competition experiments between CB-A and CB-D indicate different fates depending upon mode of bacterial growth: planktonic or biofilm, two modes in which Roseobacters, in general, and n nature , 74. We n nature . The oveS. enterica  that allow the bacterium to rapidly adapt to shifting environmental conditions [The lab and field data presented here demonstrate that both phage types can occur in mixed populations and indicate the competitiveness of a given host-virus pair is niche specific. Thus, the maintenance of both phage types within a population may be advantageous to a given host over multiple generations and across marine landscapes. Indeed, we might consider these discrete host-phage populations as analogous to bacterial populations that exhibit phase variation. Phase variation has been described as an interchange between physiological “states”, and is exemplified by the production of antigenic components, H1 and H2, in motile and nonmotile strains of nditions . Why co-nditions , 77, 78)Lysogeny is widespread in nature and has recently received considerable attention in the context of marine systems where focus has been on elucidation of the environmental factors that drive temperate phage into either a lytic or lysogenic state e.g., , 28. OurSupplemental materials