Nowadays the environment and its natural resources face many issues, related to the depletion of natural resources beside the increase in environmental pollution resulted from uncontrolled plastic waste disposal. Therefore, it is important to find effective and feasible solutions to utilize these wastes, such as using them to produce environmentally friendly green concrete. In this study, plastic-based green lightweight aggregates (PGLAs) containing PET plastic waste and by product additives were developed, and their subsequent physical and mechanical properties were compared with those of reference aggregates. Then, green lightweight aggregate concrete mixes (GLACs) were produced at 100% replacement of normal weight and lightweight coarse aggregate with developed PGLAs; and their fresh, hardened, microscopic and durability-related properties were compared to those of control mixes. Study results revealed that the unit weight of PGLAs were 21% to 29% less than that of normal coarse aggregate. Additionally, PGLAs had low water absorption that varied between 1.2% and 1.6%. The developed aggregates had 45% higher strength compared to that of lightweight coarse aggregate. Study results confirmed that structural green lightweight aggregate concretes (GLACs), that satisfied the dry density, compressive and splitting tensile strength requirements specified in ASTM C330, were feasibly produced. Finally, GLACs had low-to-moderate chloride penetration in accordance with ASTM C1202, thus it can be used in those areas exposed to the risk of chloride attack. Demand of concrete has boomed up to 30 billion ton/year in the construction industry . CurrentOn the other hand, the continuous production and use of plastic products have resulted in huge quantities of plastic  [Previously, natural coarse aggregate (CA) and fine aggregate (FA) were replaced by light weight plastic aggregates for production of lightweight concrete (LWC). For instance, LWC produced by replacing 5% to 75% FA with shredded PET bottles ,4,5,6,7.Besides decreasing the weight of the concrete, plastic aggregates significantly reduce the slump of concrete. For instance, a 95% concrete slump reduction was noticed by replacing 20% FA with plastic waste ; while aMoreover, fresh density of LWC containing plastic particles is governed by plastic proportions and type. For instance, 80% and 55% replacement of CA and total aggregates with expanded polystyrene (EPS) reduced the fresh density by 40% and 64%, respectively, ,18. SimiThe compressive strength of plastic-based lightweight concrete decrease significantly due to replacement of high compressive strength conventional CA and FA with plastic aggregates. For instance, 62% to 82% reduction in compressive strength was noticed by replacing 15% to 80% of CA with plastic  ,23,24,25In addition, previous studies reported a reduction in splitting tensile strength ranging from 26% to 38%, and from 37% to 40%, by replacing CA at 100% and FA at 75% with plastic-based aggregate , respectively. Similarly, a reduction in flexural strength that varied between 50% to 59% was reported previously at different replacement levels, which ranged from 15% to 50% of CA with plastic particles . The redIn the study conducted by Marzouk et al. , scanninOn the other hand, the durability and durability-related properties for the concrete made with plastic has received a little attention. According to Kou et al. (2009) , the chl3 and 0%, respectively [Plastic-based lightweight aggregate concrete is predominately produced by direct incorporation; however, indirect replacement, where synthetic aggregate was produced prior to its implantation in concrete, is less focused. The synthetic aggregates, such as polyethylene terephthalate (PET) and fillers , are less covered ,34,35,36ectively ,16.3 and 0% [Furthermore, lightweight aggregates were produced by melting mixed plastic comprised of 85% PET and 15% polypropylene (PP) . The pro3 and 0% . In anot3 and 0% , the syn3 and 0% .The effect of incorporating shredded or virgin plastic, as it is in concrete, has extensively been studied over the last few decades. However, less research work has been carried out to use plastic-produced synthetic aggregate in concrete. Thus, the current study focuses on the formulation of a plastic-based green lightweight aggregate (PGLA) manufactured using PET plastic waste and mineral additives. In addition, a green lightweight aggregate concrete (GLAC), comprising 100% manufactured synthetic plastic aggregates as a replacement of normal weight and lightweight aggregate, was also produced. Comparison of physical and mechanical properties for the produced green aggregates with the reference one was also made. Likewise, all the major properties including durability properties of GLACs were also examined and equated to the concrete made with conventional normal weight and lightweight aggregates.In this investigation, locally available ordinary Portland cement that complies with the standard specifications  was used. The specific gravity, initial setting time, final setting time and consistency were 3.15, 45 min, 135 min and 23.5%, respectively. The coarse aggregate was used at various levels, whilst the type and proportions of ordinary Portland cement, fine aggregates and water were constant throughout the investigation.Four different types of coarse aggregates, comprising normal weight aggregate (NCA), volcanic lightweight aggregate (VLA), Lytage aggregate (LYA) and plastic-based green lightweight aggregate (PGLA), were used in this study. The size, shape and texture of NCA, VLA and LYA are shown below in The normal weight coarse aggregate (NCA) collected from local sources in Riyadh, Saudi Arabia was used in experimental work to produce a normal weight concrete (NCAC) control mix. Additionally, a local volcanic lightweight aggregate (VLA) was used for the reference mix of local lightweight concrete (VLAC). The VLA used in this study was collected from the western side of Saudi Arabia. The LYA was supplied by Lytag International Ltd. in York, UK. The main material in this investigation was plastic-based green lightweight aggregate (PGLA), which was developed using PET plastic waste and additives, discussed in detail in The physical properties of NCA and VLA used in this study are given in However, the normal weight fine aggregate was used in the current study which comprised of 35% crushed sand and 65% red sand. The grading curves are shown ,40 in Fi3. The sample designations are provided under the table.The volume method was used in the current study for selecting the mix proportions in accordance with standard mix procedures specified in . A constIn general, the lightweight aggregate standard described in the  was used3, 0.10–0.20% and 285 °C, respectively.Due to the fact that not all plastic can be recycled, along with the differences in the physical, thermal and microstructure properties of plastic such as melting temperature ,51, a poSimilarly, three types of additives see , namely The specific gravity, absorption and unit weight of DDA and QDA were examined according to ASTM C128 and ASTM C29. Whereas ASTM C618 was followed to analyze specific gravity of FAA. The specific gravity of FAA, QDA and DDA was 2.3, 2.71 and 2.62, respectively. While the water absorption of QDA and DDA was 1.52% and 0.28%, respectively. The particle size distribution of additives was performed using the laser particle size analyzer. The median size of FAA, QDA and DDA was 6.14, 19.27 and 186.37 µm, respectively, which revealed that FAA was the finest, while DDA was the coarsest among these additives.The compression press was used in the manufacturing process of the plastic-based green aggregates. A homogenized mixture was formed, compressed, heated, melted and then cooled before grinding to give the final shape of the produced aggregates. The steps used in the production of these aggregates have already been patented under . ApplyinIn the subsequent sections, the physical, mechanical and morphology properties of the manufactured PGLA aggregates will be presented and also a comparison will be made with the NCA, VLA and LYA, along with similar studies conducted to produce plastic-based aggregates.The developed PGLAs were investigated in terms of their physical, mechanical and morphological properties. The summary of the findings of these tests are presented in The optical microscope was used to evaluate the shape and surface textures of PGLA particles. The shape of the PGLAs developed with DDA, FAA and QDA was more angular with sharp edges. This can be explained by the high degree of stiffness of PGLA, since it breaks in the crushing phase of aggregate production, rather than shredding, as reported in similar plastic-based aggregate produced in the literature.In addition, the surface textures of the PGLAs  varied fTherefore, it can be concluded that the PGLAs are comparable to that of NCA in terms of particle shape and texture. However, the PGLAs were entirely different to VLA and LYA. It was noticed that the additives type mainly governed the shape and texture of the plastic-based green lightweight aggregate. Furthermore, it was also observed that shape and texture of the SPGA series were not identical to each other, as well as that reported in the previous studies ,16,26. TThe voids between particles of aggregates are controlled by the size and shape of the particles. For example, poorly graded aggregate exhibits a higher void percentage . ConsequCategory 1—contains aggregates that diverged from the maximum limits of  for the Category 2—which diverged from the minimum limits of  for the As it can be seen in However, comparing PGLAs with NCA see  revealedThese results showed that the PGLAs made with DDA were the coarsest, followed by those PGLAs made with QDA and, finally, the finest were the PGLAs made with FAA. This is accredited to the sizes of the additive particles themselves, since the median size of DDA was the largest, whereas FAA had the smallest particles size among all the additives. The void percentage of the PGLAs see  was 13% Moreover, The specific gravity or density is a crucial property, as it is used to determine the aggregates’ volume in the mix. Additionally, controlling the density of concrete as aggregate represents almost 70% of the total volume of concrete’s components. The observed increase in specific gravity and density of PGLAs, as compared to that of VLA and LYA, is attributed to the least number of voids between PGLAs as compared to that in VLA and LYA See . HoweverFurthermore, the concretes’ quality depends on the precise calculation of aggregates’ water absorption, because some concrete properties, such as porosity, cement hydration and slump, were significantly affected with the extra or shortage of water in the mix .The results in Finally, the strength of aggregate has a direct impact on the concretes’ strength, as the stiffer aggregate provides lower impact value and vice versa . AccordiThe optical microscopic images indicate a non-uniform distribution between the plastic waste/binder (PET) and additives . These images show better bonding between the binder and additives, with fewer voids as compared to that of VLA. Additionally, these images showed the presence of voids in the PGLAs containing FAA c compareAlthough PGLAs demonstrated a significant reduction in the strength of aggregate  varying from 105 to 134% in comparison with NCA, the observed impact value of developed aggregate was less than the maximum limit  specified by . In addiThe workability of concrete mixes  was measured using the slump cone method see  in accorThe study results suggest that the maximum slump of GLACs was attained by concretes prepared using PGLA1, which was the coarsest among all developed aggregates; whereas the minimum slump was achieved by that made with PGLA2, which was the finest among all developed aggregates, as reported earlier. This can be linked with the packing level which increases with the finest aggregates and thus reduce the workability, and vice versa. The study results suggest that the concrete made with PGLA2  has potential in structural lightweight application, as it fulfils the slump requirements  given in .The fresh densities of concrete mixes containing PGLAs, NCAC and VLAC are presented in The dry density for concrete made using PGLAs is presented in Similarly, as observed in fresh density, the dry density of GLACs was slightly higher by 15 to 5% than that of VLAC. However, the dry density of the same mixes was significantly lower i.e., 14–16%) as compared to that of NCAC. The light weight of the inert materials  used to develop PGLAs was definitely the major source of the density reduction of the developed aggregates  and its concrete . This decrease is in good agreement with previous studies % as comp,27,28. IAdditionally, the minimum and maximum dry densities of GLACs were achieved by GLAC2 and GLAC1, respectively, because the aggregates used in these mixes  had the smallest and largest density/specific gravity among all PGLAs see , as repo3) by 2% to 5%, these findings were in strong alignment with those reported recently by the same author in other studies (density varied from 1906 to 1956 kg/m3) [Although the dry density of GLACs marginally exceeded the standard limits of   ,34,35,36The cube compressive strength (CS) results for GLACs are shown in The observation from this figure revealed that the increased percentage in compressive strength for GLACs varied from 3% to 14% as the curing period increased from 7 to 14 days, as compared to 16% and 18% achieved by NCAC and VLAC, respectively. Similarly, the increased percentage in compressive strength for GLACs ranged between 7% and 26% as the curing period increased from 14 to 28 days, as compared to 13% and 19% achieved by NCAC and VLAC, respectively.At 28 days, the compressive strength of GLACs varied between −8% and 13%, as compared to VLAC. However, in comparison with NCAC, the 28-day compressive strength of GLAC1, GLAC2 and GLAC3 was significantly reduced by 39%, 38% and 24%, respectively. This reduction is less prominent compared to those reported in literature ,26,28,29It worth mentioning that the highest compressive strength was achieved by GLAC3, due to the fact that the strength of the aggregate  incorporated in this mix was the highest amongst all aggregates See . OverallHowever, the 28-day splitting tensile strength of GLACs was significantly reduced from 13% to 30% and from 35% to 48%, as compared to VLAC and NCAC, respectively. These findings are in strong agreement with previous findings reported by ,16,26. IOverall, these results suggest that the GLAC3 could potentially be used in structural lightweight application since it satisfies the minimum requirements of splitting tensile strength  , as wellThe microstructure analysis of green lightweight aggregate concretes (GLACs) was evaluated using an optical microscope at 28 days, as shown in The main observation of these images indicates that the edges between the aggregates and the cement mortar are clear and well-defined, in contrast with that for VLAC. Additionally, a porous structure with internal air voids was observed for VLA particles see a. This iUnder compression loading, the mode of failure in GLACs behaved similarly to NCAC, with crack propagation through the cement matrix. In contrast with crack propagation through the aggregate itself observed for VLAC. This conclusion is in a strong agreement with , who repThe resistance of concrete containing PGLAs  towards ingressive soluble  was evaluated using a chloride ion penetration test. The results for GLACs are presented in The general trend shows that the chloride penetration of GLACs was reduced in a range varying from 43% to 72% and from 37% to 70%, as compared to VLAC and NCAC, respectively. The impermeable property of PGLAs particles  was indeed the main reason for this reduction, which; therefore, distracts the transfer of chloride ions. A similar observation was also reported by , who fouIn addition, the least permeability amongst GLACs was attained by GLAC2, which yielded a significant reduction by 72% and 70% in comparison with VLAC and NCAC, respectively. However, the highest permeability was found in GLAC1, which had less reduction by 43% and 37%, as compared to VLAC and NCAC, respectively. This variation in the permeability of GLACs is related to the level of packing in the mix , since tThese results reveal that GLACs are more resistant to chloride penetration compared to the control mixes . This is due to the closed microstructure  of the developed aggregate itself  compared with the relatively high voids accumulated in VLA and NCA, as confirmed by voids value see  and micrIn summary, these results indicate that the resistance of concrete to chloride ion penetrability increases with the incorporation of PGLAs. These results suggest a potential use for green lightweight concrete in structural and non-structural lightweight applications that are exposed to the risk of chloride attack, since it is classified as having low-to-moderate chloride penetrability according to ASTM C1202-12 .PGLAs were successfully developed using PET plastic waste and different types of by product additives. The produced PGLAs presented potential applications for the use in green concrete as a total replacement of conventional VLA and NCA, as they satisfied ASTM C330-04 standard limits. PGLAs exhibit lower water absorption and unit weight together with better grading and high strength.Green lightweight aggregate concrete mixes demonstrate high slump due to the shape and texture of the produced aggregates .The unit weight of green lightweight aggregate concretes in fresh and dry state was reduced as compared to normal weight concrete.The 28-day compressive strength of green lightweight aggregate concretes was reduced from 24% to 39%, with respect to normal weight concrete; whereas insignificant differences varied between −8% and 13%, as compared to conventional lightweight concrete.The 28-day splitting tensile strength of green lightweight aggregate concretes was significantly reduced from 13% to 30% and from 35% to 48%, as compared to lightweight and normal weight concrete, respectively.Under flexural loading, the green lightweight aggregate concretes exhibited the same mode of failure for those mixes made with conventional lightweight and normal weight aggregates, as expected. However, under compression loading, the mode of failure in green lightweight aggregate concretes behaved similarly to normal weight concrete, with crack propagation through the cement matrix; in contrast with crack propagation through the aggregate itself, which was observed for conventional lightweight concrete.The resistance of concrete to chloride ion penetrability increases with the incorporation of plastic-based green lightweight aggregates. Thus, green lightweight aggregate concrete has potential application in structural and non-structural lightweight applications that are exposed to the risk of chloride attack, as it satisfied the requirements of ASTM C1202-12.Green lightweight concrete has the potential to be used in structural and non-structural lightweight applications since it satisfied dry density, compressive and splitting tensile strength requirements specified in ASTM C330. However, these results must be checked in terms of different W/C ratio, chemical admixtures, lone term test and durability test before it can be recommended in a large-scale application.This study presents the production of plastic-based green lightweight aggregates (PGLAs) containing PET plastic waste and by product additives were developed. The physical and mechanical properties of PGLAs were then compared with those of reference aggregates. Additionally, green lightweight aggregate concrete mixes (GLACs) were produced at 100% replacement of normal weight and lightweight aggregate with developed PGLAs at W/C of 0.5. The fresh, hardened, microscopic and durability-related properties were investigated and compared to those of control mixes. The following conclusions can be drawn from this study:


The structural analysis of a 95-year-old concrete arch bridge located in Jagodnik (Poland) is performed in this paper, in order to check its behavior under today’s traffic loads. The mechanical properties of both the concrete and the reinforcement are investigated by testing cores and bar stubs extracted from the bridge. Structural analysis confirms that the bridge meets today’s load requirements in terms of bearing capacity, serviceability state, and that the adopted structural improvements (a new deck slab on top of the existing structure and a layer of mortar to protect the surface of the old concrete) are effective. In this way, the 95-year-old arch bridge was given a new life. The structural improvements show how combining numerical modelling and laboratory tests can contribute to the preservation of an old—though fairly simple—and valuable structure, otherwise destined to demolition, with both environmental and economic benefits. Arch bridges are one of the most popular types of bridges. At present, there are over 40 concrete arch bridges in the world with a span of greater than 200 m . ConcretThe present study is aimed at the structural analysis of the 95-year-old concrete arch bridge based on mechanical properties measured by means of laboratory tests. Structural analysis was a part of an expert opinion, required to check whether the old arch bridge has an adequate bearing capacity face to today’s traffic loads, in order to extend its service life. The present paper supplements and extends the investigations performed by Ambroziak et al.  on the dThe old bridge investigated see  is an arBefore the reconstruction process of the 95-year-old concrete arch bridge see  a few frThe concrete cylinders were tested in compression to the failure according to EN 12390-3 standard . ConcretThe reinforcement smooth steel bars of a 6 ± 0.1 mm diameter were taken from the old arch bridge and subjected to uniaxial tension by means of the computer-controlled Zwick Z400 testing machine , see 2). The yield strength (291 ± 7 MPa) specified in laboratory tests corresponds to the guidelines issued in the arch bridge construction time. The Post-Second World War standard PN-B-195 [2), 235 MPa (2400 kg/cm2), and 353 MPa (3600 kg/cm2). The S500 steel grade of 500 MPa characteristic yield strength is an abundant concrete reinforcement on bridge and building sites today.The Regulations on the Construction and Maintenance of Road Bridges  approvedPN-B-195  made it Additionally, two bar stubs were tested at a displacement rate equal to 10 mm/min b. The elfck,is,cycl 100 according to standard EN 12504-1 [fck,is,cube = fck,is, cycl 100 = 20.5 ± 1 MPa. The mean compressive strength of old concrete cylindrical samples slightly exceeds the value presented by Ambroziak et al. [The uniaxial compressive experimental tests were conducted on the Advantest 9 C300 KN mechanical testing machine. The experiments were performed to the failure of the concrete cylinder specimens and were used at a constant rate of loading with a range of 0.6 MPa/s according to EN 12390-3 . The uni 12504-1  are idenfck,is,cube according to EN 13791 [fm(n),is is the mean in-situ compressive strength of n = 16 test results, fis,lowest is the lowest in-situ compressive strength test results, kn is the factor depends on the number of tests results . Ambroziak et al. [The concrete had large variations in compressive strength ranging from 14.9 MPa to 29.7 MPa, see EN 13791  standardEN 13791 , s = 1 MEN 13791 , 12 MPa k et al.  in theirHigh compressive strength variation among individual concrete specimens is produced by impurities that were identified after uniaxial compressive tests, see 2) and 17.7 MPa (180 kg/cm2) for the erection of concrete structures. This guideline has been applicable till September 1925, i.e., while German standard DIN 1045 [3 aggregate in concrete mixes, see The reinforced concrete structural guideline  issued iDIN 1045  was intrDIN 1045  made it 3 concrete mix, the volume ratio of aggregates and the consistency of the ready concrete mix, see The Polish standard PN-B-195  specifie2) may be achieved by use of 400 kg of cement with a 1:2 ratio of sand to gravel parts in 1 m3 of finished concrete of a rammed concrete consistency. The standard PN-B-195 [2) with the same amount of cement. The lack of clear and detailed water dosage guidelines produced variable compressive strengths of old concrete structures. The standard PN-B-195 [2, see According to the PN-B-195 standard  the charPN-B-195  in its ePN-B-195  emphasiz3 while the median is equal to 2173.5 kg/m3, see The method specified in EN 12390-7  standard2). The modulus of elasticity assumed for investigated old concrete is 1.55 times greater than the value specified in regulations [The secant modulus of elasticity in the range 0 to 40% of the ultimate strength  under the service loads, and at the Ultimate Limit State (ULS) under the ultimate loads. The maps of bending moments in the deck slab and the concrete arch under the dead weight of the structure and loads of the bridge equipment are determined in The site inspection and inventory of old arch bridge make it possible to assess the amount of reinforcement in structural elements, see fyd) and design compressive strength of old concrete (fcd) are determined according to specified characteristic yield strength of reinforcement (fyk), characteristic compressive strength of concrete (fck), partial safety factors for reinforcement (γs) and concrete (γc) material properties. The partial safety factors γs and γc are assumed according to Table NA.2 guidelines in National Annex to PN-EN 1992-1-1 [The design yield strength of reinforcement /1000 = 6.88 mm. Determined largest deflections fulfils the SLS condition fmax = 1.32 mm < fperm = 6.88 mm.The largest displacement under the SLS combination of the traffic loadings see  is equalStructural analysis indicated that the 95-year old concrete arch bridge structure satisfies SLS and ULS requirements under the traffic loads class C according to PN-S-10030  standardAll newly erected bridges and old rebuilt bridges need a detailed assessment and dynamic analysis. In this paper, this analysis is not included but the dynamic analysis is performed to properly assess the properties of the 95-year-old arch concrete bridge. It should be noted, that improper design of bridge structure cause the risk of excessive structural vibrations throughout the operation ,71,72,73The investigated core samples of the 95-year-old concrete exhibit a high compressive strength scatter from 14.9 MPa to 29.7 MPa, see The concrete specimens tested in compression show that there is a large number of impurities in the old concrete  with cavities and soft pockets. The scatter in the mechanical properties is, therefore, due to either very low quality-control in the building phase or very poor concrete technology based on portable concrete mixers and hand-made proportioning of concrete constituents.3, see The mean dry density of the original concrete equals 2165 ± 8 kg/mfck,is,cube equals 16.9 MPa, according to EN 13791 [fcd equals 9.7 MPa.The in-situ compressive cube strength EN 13791  standardfyk = 291 ± 7 MPa) corresponds to the guidelines [The reinforced steel tensile tests confirm that the characteristic yield strength specified in laboratory tests  and SLS conditions, capable of carrying designed traffic loads class C according to PN-S-10030  in extenA 95 year-old concrete arch bridge in Jagodnik (northern Poland) is examined in this paper, from the properties of the materials—by testing samples in a lab—to the structural behavior—by finite-element modeling, with the following results:This paper provides scientists, engineers, and designers with experimental and structural assessments of the 95-year-old concrete. The study confirms the old arch bridge ability to carry newly designed traffic loads and helped give him a new structural life and extend his working life. Lots of existed old and historic bridge structures require restoration and reconstruction, which require not only a sound financial plan and the expertise of civil engineers, but—more often than not—the expertise of the scientific community, for an appropriate assessment of structural safety. Old bridge structures should return like the Phoenix from their ashes and recovery their past appearance. The protection of old bridges coincides with the preservation of their cultural heritage.


Immunoglobulin G4-related disease (IgG4-RD) is a fibroinflammatory condition. Its common manifestations include type I autoimmune pancreatitis and retroperitoneal fibrosis (RPF). We present a rare case involving a 43-year-old female who presented with left lower quadrant (LLQ) pain. Imaging of the abdomen and pelvis revealed left hydroureteronephrosis to the level of an inflammatory process in the left adnexal region, possibly reflecting a tubo-ovarian abscess (TOA). The gynecologic evaluation concluded that the mass was unlikely of gynecologic sources. Transgluteal biopsy of the mass was highly suggestive of IgG4-RD. The patient received prednisone and rituximab (RTX), resulting in complete resolution of the mass, which was confirmed on repeat imaging. This case report provides a valuable addition to the literature to highlight that the diagnosis of IgG4-RD is based on the combination of characteristic clinical, serologic, radiologic, and histopathologic findings. Also, it underlines that the management of the disease is through glucocorticoids (GCs) as the first-line agent for remission induction in all patients with active, untreated IgG4-RD. RTX therapy is an effective treatment for IgG4-RD that is refractory to GCs. Recent studies have suggested that RTX monotherapy can be used to induce and maintain remission in patients with IgG4-RD. Immunoglobulin G4-related disease (IgG4-RD) is an immune-mediated fibroinflammatory condition that can affect multiple organs. Its common manifestations include type I autoimmune pancreatitis, IgG4-related sclerosing cholangitis, and retroperitoneal fibrosis (RPF). Retroperitoneal involvement is reported in 20-56% of patients with IgG4-RD, and IgG4-RD is responsible for 57-59% of idiopathic RPF .RPF develops insidiously as the initial symptoms are nonspecific. Most common symptoms include low-back, flank, or abdominal pain, often radiating to the groin and/or side of the thigh. RPF related to IgG4-RD shows unilateral hydronephrosis in 75% of cases .Diagnosis of IgG4-RD is based on the combination of characteristic clinical, serologic, radiologic, and histopathologic findings. Glucocorticoids (GCs) are the first-line agents for remission induction in all patients with active IgG4-RD . In seleA 43-year-old female with a past medical history of uncontrolled type II diabetes mellitus, recurrent urinary tract infections (UTIs), chronic pelvic inflammatory disease (PID), abnormal colposcopy with the highest grade cervical intraepithelial neoplasia (CIN) II status post-cryotherapy, and bilateral tubal ligation (BTL) presented with worsening left lower quadrant (LLQ) pain of two months' duration. On review of systems, the patient endorsed yellow/green vaginal discharge of two months' duration without a malodor or pruritus.The remainder of the review was negative, particularly for other gynecologic, urinary, and rheumatologic symptoms including but not limited to fever, chills, weight loss, rash, Raynaud’s skin changes, photophobia, blurred vision, dry eyes or mouth, oral ulcers, joint pain, swelling or redness, dysuria, hematuria, or urinary urgency or incontinence. The patient denied any history of autoimmune disease in herself or her family. Her only home medication was metformin. Vitals were within normal limits. Abdominal examination was significant for mild LLQ tenderness without rebound or guarding. On pelvic examination, external genitalia was normal; speculum exam revealed a well-rugated vagina with thick green discharge on the vaginal walls and cervical epithelium and a friable cervix. Pap smear was performed and was negative for intraepithelial lesions or malignancy. Bimanual examination revealed an irregular, hard, smooth, nonmobile, and tender 5-cm mass on the left anterior vaginal wall starting from the left side of the cervix and extending to the pelvic sidewall that is not connected to the cervix. No cervical motion tenderness was noted. The uterus was mobile, anteverted, and non-tender with a smooth contour. Adnexa was tender bilaterally but nonpalpable. The mass was also palpable with similar characterization, as observed through a digital rectal examination.Transabdominal and transvaginal ultrasound (U/S) showed a complex left adnexal mass that could represent a left hydrosalpinx and/or tubo-ovarian abscess (TOA), left hydroureteronephrosis with normal-appearing right adnexa, and uterus with an endometrial stripe of 0.9 cm. A CT scan of the abdomen and pelvis revealed left hydroureteronephrosis to the level of a complex inflammatory process in the left adnexal region, possibly reflecting an evolving TOA with a trace amount of gas in the bladder and mild bladder wall thickening and perivesical fat stranding.Labs were significant for leukocytosis of 11.2. Urinalysis (UA) was negative for leukocyte esterase, nitrites, and white and red blood cells. The wet mount was negative. Alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen (CA) 125, B-human chorionic gonadotropin (B-HCG), chlamydia, gonorrhea, human papillomavirus (HPV), hepatitis B, hepatitis C, and HIV tests were negative. Autoimmune workup with antinuclear antibody (ANA), rheumatoid factor (RF), and anticitrullinated peptide (anti-CCP) were also negative. Blood and urine cultures returned negative as well.Given the patient's history of PID and abnormal CIN II, we consulted gynecology. They performed endocervical curettage and endometrial biopsy, which were negative for cytologic atypia or malignancy. They later performed posterior colpotomy, cystoscopy, and diagnostic laparoscopy with peritoneal biopsy, which revealed normal pelvic anatomy with healthy ovaries, surgical absence of fallopian tubes consistent with the history of BTL, and an irregular solid hard mass in the left retroperitoneal space, not communicating with the uterus or the cervix, not visualized to be impinging on the bladder, and not visible from the abdomen on diagnostic laparoscopy. Attempts to biopsy the mass under U/S guidance were unsuccessful. They concluded the mass to be unlikely from gynecological sources. Later, an abdominal and pelvic MRI was obtained, which demonstrated a retroperitoneal mass with increased enhancement adjacent to the left ovary. A CT urogram was also done, which showed a nonspecific 4.61 x 4.41-cm soft tissue density within the left adnexa that could represent TOA or a neoplasm , granulomas, or vasculitis. Serum IgG4 level was measured and found elevated at 250 mg/dL. The patient was started on broad-spectrum antibiotics for possible PID/UTI. Urology was consulted, after which a left percutaneous nephrostomy tube and left ureteral stent were placed. The patient was referred to our rheumatology clinic, after which she was started on prednisone 10 mg oral daily. One month later, she was started on azathioprine (AZT) 75 mg twice daily and received RTX 1 g IV in two doses, two weeks apart. On follow-up, the patient was found to have developed gastrointestinal symptoms likely as a side effect from AZT, which was then discontinued. She was started on methotrexate 12.5 mg subcutaneous once weekly along with daily oral folic acid. Repeat CT scan of the abdomen and pelvis about seven months after the treatment showed complete resolution of the retroperitoneal mass Figure .IgG4-RD is an immune-mediated fibroinflammatory condition that can affect multiple organs ,6, especThe hallmarks of IgG4-RD are dense lymphoplasmacytic infiltrations with a predominance of IgG4-positive plasma cells in the affected tissue, usually accompanied by some degree of fibrosis that has a characteristic "storiform" pattern (represented by a cartwheel appearance of the fibroblasts and inflammatory cells) and often by obliterative phlebitis and increased eosinophils . Serum IMost (>70%) cases of RPF are thought to be idiopathic , and theRPF develops insidiously as the initial symptoms are nonspecific. Most common symptoms include low-back, flank, or abdominal pain, often radiating to the groin and/or side of the thigh. The pain is usually dull and persistent, with no decrease at rest. Initially, non-steroidal anti-inflammatory drugs can relieve the pain, but this effect is usually temporary. In the case of involvement of the ureters, the pain can have a colic character. Patients often complain of constipation, and occasionally the involvement of the duodenum results in obstructive symptoms. Less frequently reported symptoms include scrotal swelling, hydrocele, varicocele, testicular pain and swelling, and deep vein thrombosis of the lower limbs, which are related to compression of the retroperitoneally located lymphatic vessels and veins. Entrapment of arteries can result in renovascular hypertension, intermittent claudication, or intestinal ischemia. Patients may also have constitutional symptoms such as fever, fatigue, muscle and joint pain, weight loss, and loss of appetite. Physical examination is nonspecific. In some cases, lumbar or abdominal tenderness is present, and a fibrous mass can be felt through the abdominal wall in very rare instances. The nonspecific clinical picture often significantly prolongs the time between the onset of symptoms and the correct diagnosis, leading to complications related to the advanced fibrotic process. Hydronephrosis, related to ureteral obstruction and found in 47-100% of patients, is the most frequent and the most severe form of complication. In over 50% of cases, it has a bilateral character. The frequency of renal involvement is 7-24.6% [Diagnosis of IgG4-RD is based on the combination of characteristic clinical, serologic, radiologic, and histopathologic findings. Features include mass-like swelling of involved organs, a lymphoplasmacytic infiltrate with predominant IgG4-positive plasma cells, and a variable degree of fibrosis that has a characteristic storiform pattern. The presence of these findings, often together with mild tissue eosinophilia, is strongly suggestive if accompanied by increased numbers of IgG4-positive plasma cells in the affected tissue. Depending on the type of tissues involved, the number of IgG4-positive plasma cells per high-power field (HPF) consistent with or suggestive of IgG4-RD varies. Tissue IgG4-positive cell counts and the ratios of IgG4- to total IgG-positive cells are considered secondary in importance to the histopathologic appearance of the tissue. Generally, the minimum value required for establishing the diagnosis for most tissues ranges from 30 to 50 IgG4-positive cells/HPF. However, in some organs or tissues, including the kidney and others, 10 IgG4-positive plasma cells/HPF may be sufficient. In addition, serum IgG4 levels are elevated in approximately two-thirds of the patients. Guidelines for diagnosis have been described by the Japanese Criteria, the American College of Rheumatology, and the International Consensus Guidance Statement on the Management and Treatment of IgG4-RD.All patients with symptomatic, active IgG4-RD require treatment. Some patients require urgent treatment, such as those with the disease in the pancreas, biliary tree, aorta, mediastinum, kidneys, retroperitoneum, and mesentery. Urgency is needed because uncontrolled disease in these organs can lead to irreversible damage. A subset of patients with asymptomatic IgG4-RD also requires treatment as a subclinical disease can lead to severe, irreversible sequelae. Some patients with asymptomatic IgG4-RD can be managed without treatment, e.g., asymptomatic lymphadenopathy or mild submandibular gland enlargement .Therapy for this disease has focused on GCs to date, which appears to be highly effective in induction therapy, unless contraindicated . TreatmeRTX, which is another treatment option, achieves its effects by depleting the subset of CD20-positive B cells that differentiate into the short-lived plasma cells producing the disease-associated IgG4. The treatment results with RTX in many studies strongly suggest that B-cell depletion is an effective strategy in many patients with IgG4-RD because of the strikingly targeted effect on the IgG4 subclass of IgG. Repeated treatment cycles with RTX lead to a progressive decrease in serum IgG4 concentrations, which is used as a biomarker in subsets of patients with IgG4-RD. As evidenced by our patient’s response to the treatment, B-cell depletion strategies offer a promising treatment approach toward IgG4-RD [IgG4-RD is an immune-mediated fibroinflammatory condition that can affect multiple organs. RPF is one form of its manifestations, which develops insidiously because of nonspecific initial symptoms. Symptoms of IgG4-related RPF (IgG4-RRF) include low-back, flank, or abdominal pain. IgG4-RRF shows bilateral hydronephrosis in 25% of reported cases and unilateral hydronephrosis in 75% of cases. Diagnosis involves multiple criteria and MRI might be superior to CT for the characterization of findings. RTX may be a promising therapy to induce and maintain remission in patients with IgG4-RRF.


INK4B, p14ARF and p16INK4A), our understanding of their functions and modes of action is limited to the latter two. Here, we show that in vitro p15INK4B is markedly stronger than p16INK4A in inhibiting pRb1 phosphorylation, E2F activity and cell-cycle progression. In mice, urothelial cells expressing oncogenic HRas and lacking p15INK4B, but not those expressing HRas and lacking p16INK4A, develop early-onset bladder tumors. The potency of CDKN2B/p15INK4B in tumor suppression relies on its strong binding via key N-terminal residues to and inhibition of CDK4/CDK6. p15INK4B also binds and inhibits enolase-1, a glycolytic enzyme upregulated in most cancer types. Our results highlight the dual inhibition of p15INK4B on cell proliferation, and unveil mechanisms whereby p15INK4B aberrations may underpin cancer and non-cancer conditions.Human chromosome 9p21.3 is susceptible to inactivation in cell immortalization and diseases, such as cancer, coronary artery disease and type-2 diabetes. Although this locus encodes three cyclin-dependent kinase (CDK) inhibitors (p15 The human chromosome locus 9p21.3 is a tumour suppressor hub which encodes three CDK inhibitors, p15INK4B, p14ARF and p16INK4A. Here, the authors show that p15INK4B inhibits the cell cycle and glycolysis in a murine model of HRas + ‐mediated urothelial carcinoma and has a more relevant role as a tumour suppressor than its neighbouring p16INK4A. CDKN2B gene resides on human chromosome 9p21.3 and encodes p15INK4B, a cell-cycle regulator that exhibits inhibitory activities toward cyclin-dependent kinases CDK4 and CDK6 (refs. 2). Along with CDKN2A gene, which encodes p14ARF and p16INK4A via usage of separate first exons, and signals through p53 and pRb pathway, respectively3, these three CDK inhibitors arranged in tandem on 9p21.3 constitute a prominent tumor suppressor hub that is frequently deleted or epigenetically silenced prior to immortalization of primary cultured cells6 and in pre-tumor lesions8 and a multitude of tumor types11. Unlike p14ARF and p16INK4A whose tumor-suppressive activities have been well documented, p15INK4B is often regarded as functionally equivalent to p16INK4A and a bystander during 9p21.3 deletion. These notions are, however, in stark contrast to the phenomena that (i) p15INK4B can be targeted for deletion independently of p14ARF and p16INK4A12; (ii) the three CDK inhibitors can be down-regulated separately by promoter-specific methylation, cis-acting long non-coding anti-sense RNA 15 or divergent upstream signals 17); (iii) p15INK4B becomes critically important in the absence of p16INK4A18; and (iv) in genetically engineered mice the loss of both p14ARF and p16INK4A fails to cooperate with oncogenes to elicit tumors in certain cell types, such as urothelium19. By analyzing p15INK4B in vitro, in vivo, and in human tumor cohorts, we present multiple lines of evidence here demonstrating p15INK4B as a markedly stronger tumor suppressor than p16INK4A via dual inhibition of cell cycle and aerobic glycolysis (Warburg effect)20. We provide detailed structural basis that sets p15INK4B apart from p16INK4A functionally and mechanisms whereby p15INK4B exerts its potent activities. Our findings highlight a dominant role of p15INK4B as a principal component of 9p21.3 locus in tumor suppression and the essential contributions of p15INK4B deficiency to tumor initiation. Additionally, our study reveals an entry point through which to study the elusive molecular bases of non-cancer diseases, such as coronary artery disease, type 2 diabetes, and glaucoma, all of which are prominently associated with 9p21.3 aberrations23.CDKN2B that encodes p15INK4B (p15 hereon for brevity) was because of the high frequency (up to 70%) of 9p21.3 loss in human urothelial tumors26, but the complete failure of loss of CDKN2A  to cooperate with oncogenic HRas (Q61L) to induce any urothelial tumor in mice19. We hypothesized that, instead of p14 and p16, p15 might play a more important role in urothelial tumor suppression in the context of oncogenic HRas, and that p15 loss might be more functionally reflective of 9p21.3 loss. To test this, we crossed our Upk2-HRas transgenic mice, in which the expression of oncogenic HRas was driven by urothelium-restricted murine Upk2 promoter  revealed markedly enlarged bladders ), even though the body weights did not differ significantly , albeit at a much lower frequency (30% by 10 months), later onset (beginning at 4 months) and smaller sizes . While a relatively small number of HRasWT/*/p15+/− (in which p15 was heterozygous) also developed bladder tumors, the tumor cells retained one p15 allele. We presently cannot rule out whether the remaining allele is inactivated by hypermethylation, ANRIL, or inactivation of the upstream regulators, such as TGF-β (see later). This is an important question that warrants further investigation. By histology, the p15−/− mice exhibited normal-appearing urothelium and the HRasWT/* mice urothelial hyperplasia , it was necessary that we ascertain under the same experimental condition the relative potency of p15 versus p16 (instead of p16 and p19 together) in urothelial tumor suppression as well as the in vivo effects of their loss in urothelial tumor initiation. With this in mind, we carried out crosses between Upk2-HRas* transgenic mice and p16 knockout mice in which exon 1α of CDKN2A gene was replaced with a neo gene, which inactivates p16 but not alternatively transcribed p19 . In parallel, we carried out de novo crosses between Upk2-HRas* transgenic mice and the aforementioned p15 knockout mice  HRasWT/*/p15−/− mice; and (iv) HRasWT/*/p16−/− mice . Finally, there was a strong increase of p19 expression that was confined to HRas*-expressing and p15-lacking urothelial cells  and therefore lacked endogenous p15 and p16. By first stably transfecting the cell lines with CMV-driven reverse tetracycline transactivator (CMV-rtTA) and then stably transfecting with tetracycline response element (TRE)-driven, Myc-tagged p15 cDNA or TRE-driven, Myc-tagged p16 cDNA, we were able to use tetracycline to inducibly express, detect and semi-quantify, via the Myc-tagged, restored p15 or p16. We found p15 to be significantly more inhibitory of cell proliferation than p16 in both cell lines  that had homozygous deletion of 9p21.3  with the exception of their N-termini (N) and the longer C terminus (C) unique to p16. To discern the region responsible for the strong interaction between p15 and CDK4/CDK6, we first performed domain switch between p15 and p16 by engineering p15/p16 hybrids, i.e., p15-N/p16-C and p16-N/p15-C. Transfection and inducible expression of Myc-tagged hybrids and their wild-type controls in p15/p16-lacking, parental UMUC3 cells showed that p15-N/p16-C was as effective as wild-type p15 in inhibiting pRB1 phosphorylation Fig.  and cellnts Fig.  further 40 for computational modeling and molecular dynamics (MD) simulations for p15–CDK6 interaction  and Arg168 of CDK6, we replaced Arg24 of p16 with leucine, because MD simulations of p16–CDK6 complex showed that Arg22 of p16E. coli-produced, histidine-tagged p15 as a bait, with E. coli-produced histidine-tagged p16 or histidine-tag only as controls. After coupling the bait and controls separately onto nickel columns, we incubated them with total proteins extracted from parental UMUC3 cells. The bound proteins were eluted and resolved by two-dimensional electrophoresis, after which the proteins specifically bound to histidine-p15, but not to histidine alone or to histidine-p16, were selected for mass spectrometry . We took an unbiased pull-down approach using E. coli-produced, histidine-tagged p15 and total proteins from parental UMUC3, as well as immunoprecipitation using inducible expression of Myc-tagged p15 in UMUC3 cells. Both experiments unequivocally validated the specific binding between p15 and enolase-1  along with RTK-Ras activation (~95%) in human bladder tumors41 and the lack of tumorigenicity in mice by activated RTK-Ras pathway components alone or in combination with CDKN2A deletion43. Our data strongly suggest that CDKN2B deficiency is more reflective of 9p21.3 loss and is a functionally relevant collaborative partner with RTK-Ras activation to initiate bladder tumors. Consequently, the status of CDKN2B, instead of CDKN2A, coupled with RTK-Ras activation should be used as a combinatorial biomarker set to diagnose and predict the recurrence of low-grade non-invasive bladder cancer. Because concurrent 9p21.3 inactivation and RTK-Ras activation are highly prevalent in non-bladder cancer types13, our findings call attention to the need to reexamine the effects of CDKN2B deficiency on tumorigenesis of other organ systems and cell types. Even greater attention should be paid to the tumorigenic role of CDKN2B deficiency in tumors where CDKN2B is preferentially deleted, such as familial melanoma, glioma, and certain hematological malignancies45. Second, our data reveal CDKN2B as an important player that interconnects cell cycle and glycolysis by specifically binding and inhibiting both CDK4/CDK6 and enolase-1. Thus, the deficiency of CDKN2B not only promotes tumor cell proliferation by releasing the constraints of CDKN2B on G1 to S progression, it also creates a metabolically conducive environment during tumor-induced hypoxia under which tumor cells require increased glycolysis to produce metabolic intermediates for biosynthesis of cell building blocks, such as amino acids, lipids, and nucleic acids46. By compiling gene expression data  comprising eight normal bladder cohorts and nine bladder tumor cohorts from human patients , we found enolase-1 to be significantly overexpressed in all the tumor cohorts irrespective of tumor grades, compared to the normal controls 27. Heterozygous Upk2-HRas which has been successfully maintained to date harbors a single-copy Upk2-HRas transgene and reproducibly develops simple urothelial hyperplasia of the bladder19. This line serves as breeders and a reference line representing tumor precursor lesion of the urothelium. CDKN2B (p15) knockout mouse line (a kind gift of Dr. Angel Pellicer of New York University School of Medicine) is a ubiquitous p15 knockout line in which the exon 2 of p15 gene was replaced with a neo, which abrogated p15 expression53. p16 knockout mouse line, obtained from US National Cancer Institute’s Mouse Repository, is a ubiquitous p16 knockout line in which the exon 1α of CDKN2A gene was replaced with a neo gene, which inactivated p16 but not alternatively transcribed p19 (ref. 33). Ink4ab  double knockout mouse line (a kind gift of Dr. Anton Berns of The Netherlands Cancer Institute) is a ubiquitous knockout line that was originally obtained in Dr. Berns’s laboratory by targeting p15 in the p16 knockout ES cells where exon 2 of CDKN2A bore a stop codon in the p16 reading frame but not in the p19 reading frame18. Together, this abrogated the expression of p15 and p16 without affecting the p19 expression. Intercrosses were carried out between the heterozygous Upk2-HRas line and each of aforementioned knockout (KO) lines, and the offspring was further intercrossed such that the Upk2-HRas transgene was heterozygous and the KO alleles were hetero- or homozygous for tumorigenic analyses. The Upk2-HRas transgene allele was identified by genomic Southern blotting of NcoI-digested tail DNA or by genomic PCR using specific primers  of New York University School of Medicine.Preliminary screening of hematuria, a common manifestation of bladder tumors in human patients, was performed on spot urine samples collected from conscious mice through gentle massaging of the lower abdomen. Urine samples were immediately applied atop of dipstick strips (BD Biosciences). The color change from yellow to blue on the sixth square from the top was considered positive for hematuria and the result was recorded on a digital camera.Mice of unique genotypes comprising different cohorts were sacrificed at pre-determined time points, and their urinary bladders were freshly dissected out, urine drained, weighed, turned inside out for the presence of visible tumors. The bladders were then bisected along the sagittal plane, with one half fixed in 10% PBS-buffered formalin, routinely dehydrated in serial ethanol and xylene, embedded in paraffin, sectioned, and stained with hematoxylin and eosin, and the other half scraped off its mucosa/tumors for protein preparation and biochemical work.37 were purchased from the American Type Culture Collection  and used for all transfection experiments. Both the cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM)   supplemented with 10% Tet-system approved fetal bovine serum  at 37 °C with 5% CO2.Two human bladder cancer cell lines, RT112 and UMUC3, that had 9p21.3 deletionA tetracycline-based inducible system was chosen for conditional gene expression. Briefly, plasmid pTet-on  was transfected into RT112 and UMUC3 cells using Lipofectamine 2000 , followed by selection of stable clones in 300 μg/ml G418 . pTRE2-pur containing p15, p16, p15-N/p16-C or p16-N/p15-C was then transfected into pTet-on-stably transfected RT112 or UMUC3 using Lipofectamine 2000 and stable clones were selected in the presence of puromycin (Gibco) (0.8 μg/ml for RT112 and 1.2 μg/ml for UMUC3).3) were seeded onto 96-well plates in DMEM medium with FBS containing 1 μg/ml doxycycline, and cell proliferation was determined at 24, 48, and 72 h by WST-1  with a 96-well plate reader at 450 nm absorbance .Cultured bladder cancer cells  and sorted with a FACSCalibur analyzer, and the data were analyzed by FlowJo software (FlowJo 10.4.2).Cultured cells were detached by trypsin digestion, collected, and washed by centrifugation at 800 × E2F activity was assessed by transient transfection of UMUC3 cells with an E2F-luciferase reporter construct . Briefly, UMUC3 cells that reached 60% confluence were transfected the E2F reporter plasmid in the presence of Lipofectamine 2000 . After 12 h of incubation, the transfected cells were lysed, supplemented with dual-luciferase reporter assay kit (Promega), and the luciferase activity was measured using a luminometer .40 was used as a template to model the molecular interactions between p15 and CDK6. The existing p16–CDK6 model missed two structural parts: one in CDK6 (residues 49–71) and the other in the N terminus of p16 (residues 1–9) 55 was used as the template to build the missing part 40 and p15 from p15 NMR ensemble (PDB id: 1d9s)58. Among the five p15 models predicted by I-TASSER based on p16, the one with the highest C-score was selected and superimposed on the p16–CDK6 complex to build p15–CDK6 complex 60. AmberTools61 were used to create topology and coordinate files for the structures. Each system was neutralized by adding counterions of Na+ or Cl−, and was solvated in rectangular water box full of TIP3P water, so that the boundary of the box is at least 12 Å away from any solute atom. The cutoff distance was set at 10 Å for short-range Coulomb and van der Waals interactions, and the particle mesh Eward (PME) method62 was utilized to treat long-range electrostatic interactions. The SHAKE algorithm63 was employed on all atoms covalently bonded to hydrogen atom, allowing for an integration time step of 2 fs. The simulation detail is as follows. Each system was minimized by three steps: (i) the system was minimized by 3000 steps of the steepest descent minimization followed by up to 2000 steps of conjugate minimization, and harmonic restraints with a force constant of 5 kcal/mol Å were applied to all solute atoms; (ii) the above minimization procedure was repeated and harmonic restraint with a force constant of 1 kcal/mol Å imposed on solute atoms; and (iii) no harmonic restraint was imposed on solute atoms during the minimization. After minimization, each run was subjected to equilibration in following steps: (i) 50 ps constant volume ensemble (NVT) MD simulations with 2 kcal/mol Å restraints on the solute atoms, and the system was gradually heated from 10 to 300 K. (ii) 50 ps NVT MD at 300 K and with 2 kcal/mol Å restraints on the solute atoms. (iii) 100 ps isothermal isobaric ensemble (NPT) MD at 300 K and with 0.5 kcal/mol Å restraints on the solute to adjust the solvent density. Finally, production MD simulations were carried out for 150 ns at a constant temperature of 300 K and a constant pressure of 1 atm. The temperature and pressure were maintained using Langevin thermostat and Berendsen barostat methods, respectively. The atomic coordinates of the complexes were saved every 100 ps to obtain the trajectories for post structural analysis.Two comparable systems: p16–CDK6 and p15–CDK6 complexes as well as an additional p16(R24L)–CDK6 complex were investigated in this study. For each system, five independent MD replicas were carried out using same starting structural configuration, but with different initial velocities. Total simulation time of 2.25 μs was performed  were aligned and clustered. The clustering was performed on the binding interface residues by using the DBSCAN method64 with min-points of 50 and epsilon of 1.2 Å, which is implemented in cpptraj module of AmberTools61. For the p15–CDK6 system, nine clusters were generated and the most populated one (832 out of 5000 snapshots) was selected for p15–CDK6-binding interface analysis  profiles of protein C alpha atoms  by PCR using Q5 High-Fidelity DNA Polymerase (New England BioLabs), with p15- and p16-specific primers  (1-methionine to 51-isoleucine of p15/52-glutamine to 156-aspartate of p16) and another comprising the N terminus of p16 and the C terminus of p15 (p16-N/p15-C) (1-methionine to 49-isoleucine of p16/51-isoleucine to 138-aspartate of p15) were constructed using the following procedures. First, PCR was carried out using pTRE2pur-p15-Myc as a template using primer 1 (see Supplementary Table E. coli (New England BioLabs) to produce recombinant His-tagged p15 and His-tagged p16 after the induction by IPTG. The His-tagged proteins were purified on Ni-NTA Superflow Columns (Qiagen).Histidine (His)-tagged p15 and -p16 were amplified using pTRE2pur-p15-Myc and pTRE2pur-p16-Myc as templates, respectively, by PCR using Q5 High-Fidelity DNA Polymerase (New England BioLabs). The primers for His-tagged p15 and His-tagged p16 are listed in Supplementary Table The N-terminal deletion fragments of p15 cDNA were generated by PCR using wild-type p15 cDNA as template with Q5 Site-Directed Mutagenesis Kit (New England BioLabs), with specific primers for Δ2–15, Δ2–27, Δ2–37, and Δ2–49 as listed in Supplementary Table E. coli transformation, respectively. The mutagenesis primers for p15/E16A, p15/S20A, p15/R24A, and p15/R45A are listed in Supplementary Table Point mutations were created using site-directed mutagenesis in p15 using two sets of plasmids as templates, i.e., pTRE2pur-p15-myc-tag and pET28a-p15-his-tag, which were used for bladder cancer cell line transfection and Cultured UMUC3 bladder cancer cells were lysed in RIPA buffer . The soluble proteins were incubated with mouse anti-Myc-tag (Supplementary Table E. coli/BL21 with gentle rotation at 4 °C overnight. The mixture was loaded onto Ni-NTA Superflow Columns (Qiagen), incubated and washed, and the bound proteins were eluted with different concentrations of imidazole solution. Western blotting was performed to detect the proteins in each eluted fraction. The bound proteins on western blotting from both immunoprecipitation and pull-down experiments were semi-quantified by scanning the protein bands using NIH ImageJ software.Total protein lysates were prepared from cultured UMUC3 cells in RIPA buffer and then incubated with His-tagged proteins produced in p15 protein purchased from Abcam was dissolved in 100 µl (2.5 μg/ml) coating buffer  and pre-coated onto a clear flat-bottom 96-well plate (Thermo Fisher Scientific) at 4 °C overnight. After washing three times with PBS, the pre-coated wells were blocked with 1% bovine serum albumin (BSA) (W/V) in PBS at 37 °C for 1 h, after which the wells were washed three times with PBS and supplemented with CDK4 (5 μg/ml in 100 μl) (Novus) in the presence of 0–40 μg/ml of enolase-1 . Following additional incubation at 37 °C for 2 h, the wells were washed in PBS containing 0.05% v/v Tween-20 and incubated with 100 μl rabbit anti-CDK4 antibody  was reengineered to bear an shRNA of enolase-1 (GCATTGGAGCAGAGGTTTACC-TCAAGAG (loop)-GGCGTTCAATGTCATCAATGG). The lentivirus was packaged in 293 T cells by co-transfection with the plasmids, VSV-G, PAX2, and the shRNA-bearing vector. The packaged lentivirus was then used to transfect UMUC3 cells harboring Myc-tagged p15, and the stable transfected cells were selected in the presence of 4 μg/ml of blasticidin (InvivoGen). Co-immunoprecipitation of p15 with CDK4 and CDK6 as well as cell cycle and cell proliferation analyses were described above.E. coli that served as baits were separately immobilized on Ni-NTA Superflow Columns and incubated with total proteins extracted from parental UMUC3. After washing, the bound proteins were eluted with imidazole. Two-dimensional electrophoresis was performed based on the carrier ampholyte method of isoelectric focusing66. Briefly, the first dimension  was performed using 2% pH 3–10 Isodalt Servalytes . Tropomyosin  served as an internal standard. After equilibration in 10% glycerol, 50 mM dithiothreitol, 2.3% SDS, and 0.0625 M tris, pH 6.8, the first-dimensional gel was loaded atop a 10% SDS-PAGE, with molecular weight standards : myosin , phosphorylase A , catalase , actin , carbonic anhydrase , and lysozyme . Protein spots that were not present in the control or p16-bound fractions were excised, in-gel trypsin-digested, HPLC-purified and subjected to mass spectrometry following established procedures67.Recombinant proteins ) produced in 6) were lysed with ice-cold Enolase Assay Buffer . The enolase activity and standard curve were both determined according to the instructions of enolase activity colorimetric assay kit (Biovision).Cultured bladder cancer cells  at 3 × 104 cells/well density in 500 µl XF Base DMEM medium  and incubated at 37 °C in 5% CO2. The assays were conducted at different time points by adding glucose, Rot/AA, and 2-DG successively in an XFe24 extracellular flux analyzer , and the ECAR data were recorded automatically.Urothelial cells were harvested from the mucosal surfaces of the inside-out mouse bladders. The cells were incubated in a Liberase solution at 37 °C for 15 min, after which the digestion was stopped by adding 2% fetal bovine serum and 0.2 μM EDTA. The cells were then cultured in cell culture microplates pre-coated with poly-For staining the de-paraffinized sections were subjected to antigen retrieval by microwaving at the maximum output in antigen unmasking solution  for 20 min. After incubation in 4% Triton X-100 in PBS, the non-specific sites were blocked with 1% BSA in PBS. The primary antibodies used for immunofluorescence staining and their dilutions are listed in Supplementary Table For western blotting, cultured human bladder cancer cells or mouse tissues were lysed in whole-cell lysis buffer containing 20 mM Tris, pH 7.6, 50 mM NaCl, 10% SDS, protease, and phosphatase Inhibitors . The protein concentrations were determined by Pierce BCA Protein Assay Kit (Thermo Scientific), and 30 μg of the proteins each sample were resolved on SDS-PAGE. The proteins were then electrophoretically transferred onto PVDF membrane, blocked with 5% BSA in TBST, and incubated with the primary antibodies with proper dilutions (Supplementary Table t-tests (two-sided), and differences for the tumor-free rates were analyzed using the Kaplan–Meier method followed by log-rank statistical test using software SPSS v17.0. Differences were considered significant when P value is less than 0.05.Data were shown as mean ± SD from at least three separate experiments and/or at least three biologically independent samples. Differences between two data sets were determined by Student Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


The COVID-19 pandemic has severely affected healthcare delivery across the world. However, little is known about COVID-19’s impact on home healthcare (HHC) services. Our study aimed to: (1) describe the changes in volume and intensity of HHC services and the crisis management policies implemented; (2) understand the responses and the experiences of HHC staff and clients. We conducted an explanatory sequential mixed methods study. First, retrospective client data  from four Dutch HHC organizations was analyzed. Second, four focus group interviews were conducted for the strategic, tactical, operational, and client levels of the four HHC organizations. Our results showed that both the supply of and demand for Dutch HHC decreased considerably, especially during the first wave (March–June 2020). This was due to factors such as fear of infection, anticipation of a high demand for COVID-19-related care from the hospital sector, and lack of personal protective equipment. The top-down management style initially applied made way for a more bottom-up approach in the second wave (July 2020–January 2021). Experiences vary between levels and waves. HHC organizations need more responsive protocols to prevent such radical scaling-back of HHC in future crises, and interventions to help HHC professionals cope with crisis situations. The COVID-19 pandemic has led to radical changes in healthcare delivery across the world since its onset in the beginning of 2020  how a client could become more independent, but not every client is open to that. Now [during the pandemic], clients were much more receptive.” (manager A2a)“A few weeks after care was scaled back, we had informal caregivers contacting us saying: ‘I said I could take over [the care for the client], but I am having a tough time’.” (director D1a)particularly by clients with cognitive decline. Some other significant adverse effects of the pandemic and the associated care restrictions were reported mainly by the nurses and the members of the client councils. As the scaling back of HHC took place at the same time as many other restrictions in the social environment—such as the closure of daycare facilities, restrictions on social contact, and so on—this led to more loneliness among clients. However, according to all the nurses, for some clients the reduction in formal care  led to undertreatment, eventually resulting in an even higher need for HHC. The consequences of limited formal care were felt “ the daily routine for clients with dementia has changed, making them more depressed, confused, and lonely. That does not necessarily mean that they need to be admitted to a nursing home, but we are doing a lot to help these clients”. (district nurse B2b)top-down manner to start with, creating a crisis team to bring together high-level management functions, including directors, operational managers, and policy advisors. For managers, communicating with HHC professionals was difficult. This was because they had to work from home and thus felt more distant from HHC professionals. It was also hard to structure the very large amounts of information they received from within and outside of their organization. Across the HHC organizations studied, for managers, the first wave of the COVID-19 pandemic was an unprecedented crisis that unfolded at great speed. Communication between HHC organizations intensified, and active regional collaboration was initiated. Directors and managers very much valued this sense of solidarity between organizations. Internally, all organizations responded in a “HHC professionals couldn’t see the wood for the trees, and neither could we as managers when we were getting 80 e-mails just about ever-changing COVID-19 policies.” (crisis team member C2a)scale back regular care to the absolute minimum in anticipation of massive COVID-19-related demand for care from the hospital sector. Due to the high demand for COVID-19-related hospital care, hospitals were already scaling back planned non-COVID-19-related care, such as minor surgery, leading to a decrease in post-hospital HHC.Of the many policy decisions made by the crisis team, two were raised the most frequently and discussed in the most detail during the focus group interviews. First, the decision to In the first wave, the needs of all (regular) HHC clients were re-assessed and subsequently categorized depending on how essential their care needs were (known as the ‘traffic light system’ by some organizations). This meant that essential, medically necessary care—such as palliative care and technical nursing care —had to be continued under all circumstances. Other types of care—such as personal care, preventive home visits—were categorized in terms of necessity and alternative forms of care provision were suggested. Examples include teaching an informal caregiver to give a client’s eye drops, decreasing the frequency of showering or washing from three times to once a week, or regular telephone contact to check how a client was doing. The re-assessment of care needs and types of care went hand in hand with a rise in the use of e-health tools and assistive equipment in three out of the four HHC organizations.“In the first wave, we had no idea what to expect, so we developed and introduced scenarios for scaling back, because we thought we might not have enough HHC personnel available. But actually the situation wasn’t as bad as expected for us.” (crisis team member C2a)“For clients whose care provision was scaled back, our organization made sure they had weekly telephone contact with them to support them as well as possible.” (client council member B4b)The end of the first wave was characterized by a fall in the number of COVID-19 cases. According to the directors, it became clear in hindsight that the scaling back of HHC during the first wave had been too rigorous. HHC organizations learned from the first wave, resulting in much less scaling back of HHC services during the second wave. This was in line with our quantitative findings i.e., , in whic“Based on the numbers [of infections] in our region, we expected a huge wave of clients [to be discharged from the hospital] who would need HHC. From all sides, we were being told: ‘Get ready for [clients coming out of the] hospitals, scale back your care!’ However, this huge wave never came. […] Looking back, we scaled back more than was strictly necessary, but nobody knew that at the time. […] You assumed that [the need to scale back care] would only last for a few weeks, and that we’d manage it.” (director D1a)limited information and resources available to protect nurses in the HHC setting was addressed. In the first wave, there was a lack of knowledge and experience within HHC organizations and among clients regarding what COVID-19 was and how to work with COVID-19-infected clients. Clients (suspected of) having COVID-19, therefore, received HHC separately from other regular clients, from special COVID-19 teams. For most organizations, nurses could join the COVID-19 teams voluntarily. During this first wave, HHC organizations had to deal with a severe shortage of PPE, such as facemasks and gloves. According to national guidelines, HHC professionals were not supposed to use PPE with all HHC clients, but only when visiting a client infected with COVID-19. Policy advisors and directors of the HHC organizations found themselves in a dilemma: either stick to the national policy—which most of them did—or make their own decisions. For example, some managers indicated that they would sometimes disregard national policy in individual cases, using their own professional insight. Some HHC organizations also tried to purchase extra equipment themselves rather than waiting for action at the national level.Second, the role and responsibilities of management in the “[Our HHC professionals] felt extremely unsafe, and there was nothing we could do about it because the resources just weren’t there. […] As a manager, I found that very difficult.” (manager A1a)“At a certain point, we [our organization] bought PPE ourselves because we weren’t getting anything from the regional distribution of equipment. […] That degree of divergence between organizations—that shouldn’t be allowed to happen in my opinion.” (director C1a)Since the second wave began, PPE became more widely available again and all professionals were able to wear the equipment when this was considered necessary. This—together with increased knowledge on how to live and work with COVID-19 and the fall in the number of cases—made it possible to return COVID-19-related care to the regular HHC teams. This also fulfilled the need for HHC organizations to implement a longer-term solution than COVID-19 teams.professional expertise to become an integral part of the decision-making processes and limited their autonomy in care provision. During the pandemic, nurses functioned as a link between management and clients. They had to communicate decisions on crisis management policies made by management to the individual clients. This was to provide personalized information and avoid unrest among the clients as much as possible. Due to the overall discontent with the top-down approach and the nurses’ central role, most organizations added nurses to the crisis team—and client council members too—to ensure a more bottom-up approach in the second wave.On the one hand, the top-down approach in the first wave was generally viewed positively by both managers and nurses because it enabled fast decision making. However, nurses also indicated that it did not allow for their “We received feedback from our employees that they felt there was a major gap between them and their managers in the first wave. So in the second wave, […] we introduced all sorts of initiatives for our employees to be more involved in decision making on our policy.” (manager A2a)heavy workloads during the pandemic. Particularly those working in the COVID-19 teams, but also nurses in teams where more colleagues were absent due to sickness, for example, worked longer hours than normal (some nurses mentioned 15 h working days) and were unable to take any days off. Nurses in regular HHC teams also faced additional tasks at work due to the scaling back of care in other settings such as hospital care and nursing homes, arranging COVID-19 tests for their HHC clients, or re-organizing schedules due to colleagues who were sick. Nurses experienced “I received many phone calls in my own time, from colleagues asking for help or telling me about clients who were infected. […] I was constantly thinking about who was or might be infected, who could come out of quarantine, etc. […] And arranging for clients to be tested for COVID-19 by their GP took up a lot of my time.” (district nurse D3a)working environment was considered very unsafe by nurses. According to nurses, their organizations were following national policy guidelines on PPE for much too long, and they should have been doing more to protect their employees. Many nurses and other HHC professionals felt let down by the government and their employers. Nurses were afraid of getting infected and spreading the virus to clients or to their own families. This also led to frequent testing for the virus, which further added to the perceived stress. In addition to the unsafe working environment, the information overload from managers and continuously changing procedures and protocols led to unrest among nurses, especially during the first wave. Nurses were not always aware of the latest procedures and protocols, which sometimes affected clients negatively. During the second wave, although more knowledge and PPE were available and changes in procedures and protocols were less frequent, the return of COVID-19-related care to the regular HHC teams increased uncertainty among the nurses once again. Good dissemination of knowledge from the COVID-19 teams to the regular HHC teams and the availability of experts on COVID-19-related care to answer questions were helpful in these cases, and reduced levels of anxiety and uncertainty. Even though the working conditions were considered tough by nurses, they also acknowledged the difficulties that managers and directors must have been experiencing. During the first wave, the “As employees, we were obliged to go to work, even if you had a family member at home who had tested positive for COVID-19. I felt pressure because of that.” (district nurse D3a)“[The level of unrest during the second wave] was different in each team. Some teams said: ‘We have PPE now. We know who has tested positive and what to do.’ But other teams still panic a little if a client tests positive, wondering ‘What should we do now?!’” (policy advisor B2a)emotionally impacted by these difficulties, especially those who had a high number of sick colleagues in their teams. While in the first wave, nurses were able to soldier on, their resilience by the end of the second wave was felt to be just enough to carry on. The uncertainty around future waves of COVID-19 was putting a strain on all those involved in HHC. Nurses, as well as directors, managers, and clients, wondered if people could cope with the COVID-19 situation any longer. Monitoring long-term effects, which are to be expected, at a personal level would be a valuable exercise.Nurses indicated that their resilience and wellbeing had been negatively impacted by the difficult working conditions and the difficult ethical choices that had to be made when scaling back HHC provision to clients in need. Nurses were and continue to be “Some of my colleagues believe that they infected clients. They still have that on their mind and it’s a source of stress, and as a result they are currently on sick leave.” (district nurse C3b)“ the capability to carry the burden [of the COVID-19 pandemic] was lower compared to the first wave, but we all still just got on with it.” (manager A1a)“When I see how everything is going now, I wonder how the next wave will turn out. […] [The HHC organizations] made it through the second wave, but whether it will continue to work and whether they have the resilience to absorb the next hit… I find that a frightening thought to be honest.” (client council member D4a)The main findings of our research are that particularly during the first wave of the COVID-19 pandemic , both the demand for and supply of HHC decreased considerably. The different perspectives in the focus group interviews enabled us to understand these changes in light of the responses to and experiences of HHC staff and clients to the COVID-19 pandemic in 2020. Both clients newly eligible for HHC and existing clients opted out of formal care and relied (more) on self-care and informal care. At the same time, HHC organizations reduced the provision of care in anticipation of very high demand for COVID-19-related care from the hospital sector and a lack of PPE. During the period of reduced supply, nurses monitored their clients and adjusted the supply of HHC on the basis of clients’ needs. During the first wave, HHC organizations used a centralized, top-down approach to cope with the unanticipated crisis. After the first wave, HHC organizations granted more professional autonomy to nurses. Additionally, HHC organizations learned lessons from the first wave with regard to scaling back care, making smaller reductions in HHC services during the second wave compared to the first wave. Experiences of working in HHC during the COVID-19 pandemic differed between participants and between waves. While there was a prevailing sentiment of ‘we survived’, there were also fears about how to deal with potential future waves.We saw the use of HHC decline significantly across the board between March 2020 and January 2021, with the most significant decrease in production occurring during the first COVID-19 wave. While all HHC organizations participating in our study scaled back care in a structured manner, our findings suggest that in retrospect the steps taken were ‘too much, too soon’. Although we do not know what proportion of the reduction in the use of services was due to clients preferring to avoid medical care, this is likely to have been a significant factor. However, based on our focus group interviews, one overarching factor clearly influenced all stakeholders : fear. Similar to our findings, studies in other countries suggest that many clients—and/or their informal caregivers—were unwilling to allow HHC professionals into their homes during the first wave of the pandemic because they were so concerned about being infected . At the There was an expectation that service levels might remain at a lower level after the reduction in HHC because of clients’ increased self-reliance; however, our findings suggest that has not happened. As in other studies , serviceThere are widespread reports of increased stress, burnout, and mental health problems among healthcare professionals caring for COVID-19 patients, including depression, insomnia, and post-traumatic stress disorder ,22,23. SAdditionally, decisions regarding the discontinuation of care cannot be taken on the basis of a one-size-fits-all approach and the expertise of district nurses should be utilized if we are to prevent adverse outcomes for vulnerable and already disadvantaged populations of HHC clients . It seemFinally, although telehealth has been described as a potentially valuable tool for enabling HHC visits without exposing clients and nurses to the risk of COVID-19 infection, our participating organizations turned to a simpler solution in order to maintain contact with vulnerable clients: phone calls. A recent study in the US found that a phone-based outreach program during the COVID-19 pandemic is a relatively simple intervention with a potentially far-reaching consequences . It faciThe strength of our study was its combining of quantitative data with in-depth focus group discussions that included a range of perspectives—including nurses, management, and clients—by studying a large amount of quantitative client data from four organizations. First, we were able to obtain feedback from those organizations and direct the discussion during the focus group interviews. Although we believe that the HHC organizations involved in this research are representative of the Netherlands, and only minor differences were noted between organizations in the focus group interviews, we cannot exclude the possibility that other organizations may have responded differently to the crisis. We must, therefore, be cautious regarding the generalizability of these results. A further limitation of our study is the relatively short timeframe in which it was conducted, especially given that the pandemic is still ongoing. It would be interesting to investigate the longer-term effects of the COVID-19 pandemic on HHC. Future research would therefore be valuable, in which possibly other quantitative analyses are applied on a more extensive dataset of, for example, multiple years, to assess these effects. However, we believe that the insights that we have gained into the first and second waves provide valuable lessons for future crises and for subsequent waves in this pandemic.This study shows that HHC organizations had to deliver HHC to vulnerable clients in difficult times during the first two waves (March 2020–January 2021) of the COVID-19 pandemic. In retrospect, HHC organizations scaled back the delivery of HHC too drastically in the first wave. In the future, more responsive crisis management protocols should be made available that scale back care only if this is truly needed. Nurses monitored and adjusted the provision of HHC on the basis of individual needs. However, future research will need to demonstrate whether certain HHC clients were particularly impacted by the scaling back of HHC and for which groups self-care, informal care, or e-health solutions might be appropriate. Notwithstanding all the efforts to provide the best HHC possible tailored to clients’ needs, if those providing the care do not receive sufficient support, there is nobody else to act during times of crisis. Developing and implementing interventions to help HHC professionals cope with the effects of a crisis is therefore essential for the long-term sustainability of high-quality HHC.


Biological distribution was assessed in control and lung-infection-model mice with EC-14 using 3H-L- and D-Met, and 18F-FDG. A maximum accumulation of 3H-L- and D-Met, and 3H-L- and D-Ala occurred in the growth phase of EC-14 in vitro. The accumulation of 3H-L-Met and L-Ala was greater than that of 3H-D-Met and D-Ala in both EC-14 and HaCaT cells. For all radiotracers, the accumulation was greater in EC-14 than in HaCaT cells at early time points. The accumulation was identified at 5 min after injection in EC-14, whereas the accumulation gradually increased in HaCaT cells over time. There was little difference in biodistribution between 3H-L-and D-Met except in the brain. 3H-L- and D-Met were sensitive for detecting areas of infection after the spread of bacteria throughout the body, whereas 18F-FDG mainly detected primary infection areas. Therefore, 11C-L- and D-Met, radioisotopes that differ only in terms of 3H labeling, could be superior to 18F-FDG for detecting bacterial infection in lung-infection-model mice.The effectiveness of L- and D-amino acids for detecting the early stage of infection in bacterial imaging was compared. We evaluated the accumulation of  Staphylococcus aureus and fluoroquinolone-resistant Escherichia coli fluoro-D-glucose (18F-FDG) and [S-methyl-11C]-L-methionine (11C-L-Met), radiotracers in clinical use for tumor imaging [11C]-methylaminoisobutyric acid ([11C]MeAIB), a chemical analog of alanine (Ala) in human tumor cells -L-Met (3H-L-Met), [S-methyl-3H]-D-Met (3H-D-Met), -L-Ala (3H-L-Ala), and -D-Ala (3H-D-Ala)  were used in the in vitro studies. 2-Deoxy-2-[18F]fluoro-D-glucose (18F-FDG), which was synthesized at our PET facility, was used in the in vivo biological distribution study.[S-methyl-Escherichia coli EC-14  was isolated as the clinical isolate strain. In pre-cultivation, EC-14 stock solutions with 50% glycerol were mixed with THY medium consisting of Todd–Hewitt Broth  and 0.2% yeast extract  in the ratio of 1:100 and incubated at 37 °C for 12–14 h with shaking. After incubation, EC-14 (1.2 × 108 CFU/100 µL) was seeded in Dulbecco’s modified Eagle’s medium , which does not contain amino acids (amino-acid-free DMEM) and incubated at 37 °C with shaking, for the experiments. Bacterial protein was measured using the PierceTM BCA Protein Assay Kit .2.The human keratinocyte cell line   was usedE. coli EC-14 at 1.2 × 108 CFU/100 µL were seeded in 5 mL of amino-acid-free DMEM and incubated for 1, 2, 4, 6, 8, 12, and 24 h. After incubation, 37 kBq/10 µL of 3H-L-Met, 3H-D-Met, 3H-L-Ala, and 3H-D-Ala were added to the bacterial solution and incubated for 5, 30, and 60 min at 37 °C with gentle shaking. After conducting this time-course experiment in which the bacterial incubation time was varied, we investigated the accumulation of 3H-L-Met and 3H-D-Met in each of the E. coli EC-14 and HaCaT cells, for various incubation times after 3H-L-Met and 3H-D-Met were added. EC-14 was incubated in amino-acid-free DMEM for 4 h. EC-14 was then collected by centrifugation at 7000× g for 10 min at 4 °C, washed three times with 5 mL of phosphate-buffered saline , and lysed by 1 mL of 0.1 M NaOH. Five hundred microliters of bacterial lysate was mixed with 5 mL of ULTIMA GOLD  and the radioactivity of the mixture was measured using a liquid scintillation counter . In measuring the incorporation rate of 3H-L- and D-Met into the protein fraction, another 190 µL of the bacterial lysate consisting of 0.1 M NaOH was mixed with 10 µL of 100 w/v% trichloroacetic acid . After mixing, the precipitated protein was collected on a glass fiber filter , washed three times with 1 mL of ice-cold 5% trichloroacetic acid solution, and fixed by heating at 100 °C for 1 h. The fixed protein was mixed with 5 mL of ULTIMA GOLD and the radioactivity was measured and evaluated as the incorporation rate into the protein fraction.4 per cell in a 24-well plate in DMEM and cultured at 37 °C in an atmosphere of 5% CO2. For 3H-L-Met, 3H-D-Met, 3H-L-Ala, and 3H-D-Ala accumulation assay, DMEM was removed and 450 µL of amino-acid-free DMEM was added to the HaCaT cells followed by incubation at 37 °C for 10 min. We then added 37 kBq/50 µL of 3H-L-Met, 3H-D-Met, 3H-L-Ala, or 3H-D-Ala to the HaCaT cells, which were incubated at 37 °C for 5, 30, and 60 min with gentle shaking. After incubation, the cells were washed twice with 500 µL of ice-cold amino-acid-free DMEM and lysed by 1 mL of 0.1 M NaOH. Five hundred microliters of cell lysate was mixed with 5 mL of ULTIMA GOLD and the radioactivity of the mixture was measured by LSC-5100. The incorporation rates of each radiotracer into the protein fraction were measured the same as that described in the section “3H-L- and D-Met in E. coli EC-14”.Accumulation of HaCaT cells were seeded at 5 × 10g for 5 min at 4 °C and suspended in amino-acid-free DMEM for inoculation to mice. Jcl:ICR mice  were purchased 7 days prior to infection and treated intraperitoneally with 150 mg/kg and 100 mg/kg of Endoxan (Shionogi) at 4 days and 1 day prior to infection, respectively. The lungs of mice were infected with EC-14 at 3–7 × 107 colony forming units (CFU)/80 µL under isoflurane  anesthesia. At 1, 2, 4, 8, and 24 h after infection, the infected mice were euthanized with isoflurane and the lung tissue was collected and homogenized with PBS. CFUs in homogenized solution were measured by serial dilution in PBS and plating on LB agar plates.All applicable institutional guidelines of Kanazawa University for the care and use of animals were followed. All procedures were performed in accordance with the ethical standards of Kanazawa University  and with international standards for animal welfare and institutional guidelines. EC-14 was pre-cultured in Luria–Bertani (LB) broth  for 12 h followed by seeding into new LB broth in the ratio of EC-14 solution (LB broth = 1:100) and incubation at 37 °C for 12–14 h with shaking. After pre-incubation, EC-14 was collected by centrifugation at 8000× 7 CFU/80 µL was infected to the lung of immunosuppressed mice, as described in the “E. coli EC-14 lung-infection-model mice” section. At 4 and 24 h after infection, 50 kBq/50 µL of 3H-L-Met and 3H-D-Met, and 17 MBq/90 µL of 18F-FDG were administered intravenously to the mice (n = 4), which were fasted for 4–5 h prior to administration. Mice were euthanized at 15 and 60 min after administration and the blood, heart, lung, liver, spleen, pancreas, kidney, brain, bladder, infected lung, and contralateral noninfected lung were collected. Organ samples were adjusted to approximately 100 mg and mixed with 1 mL of ULTIMA GOLD, followed by the initial measurement of 18F-FDG radioactivity using a γ-counter . The samples were incubated overnight at room temperature, mixed with 3 mL of ULTIMA GOLD and 100 µL of hydrogen peroxide, and then incubated for 1–2 more days. After incubation, the radioactivity of 3H-L-Met and 3H-D-Met was measured by LSC-5100.EC-14 at 3–7 × 10p values were calculated using the two-tailed unpaired Mann–Whitney t-test for comparison between two groups using GraphPad Prism 8 statistical software . A p value less than 0.01 or 0.05 was considered to indicate statistical significance. Data are presented as means and standard deviation. 3H-L-Met, 3H-D-Met, 3H-L-Ala, and 3H-D-Ala in E. coli EC-14 at 5 min after each injection are shown in Figs 1 and 2. 3H-L-Met and 3H-D-Met accumulation was greatest at 4 h after incubation . The accumulation of 3H-L-Met and 3H-L-Ala was greater than that of 3H-D-Met and 3H-D-Ala in both EC-14 and HaCaT cells. In both D-amino acids, the accumulation was higher in EC-14 than in HaCaT cells. Avid accumulation was identified at 5 min after 3H-L- and D-Met and 3H-L- and D-Ala injection in EC-14, whereas the accumulation gradually increased in HaCaT cells over time. In EC-14, the proportion of accumulation of 3H-L-Met and 3H-L-Ala into the protein fraction was similar to that of 3H-D-Met and 3H-D-Ala. Human cells generally take in L-amino acids and then synthesize proteins to produce energy but take in few D-amino acids [11C-MeAIB, which is an analog of Ala, showed little accumulation in an in vitro study with EC-14 (no data), possibly because of the slight difference in its chemical structure compared with Ala. For this reason, we did not use 3H-L- or D-Ala in our biological distribution study. We compared the accumulation of no acids . In contno acids ,20,21, ino acids . TherefoMuscle-infection-model mice have generally been used in bacterial infection imaging studies. However, we used lung-infection-model mice  for the E. coli and HaCaT cells could be applied to bacterial imaging. Using lung-infection-model mice, we conducted a biological distribution study in EC-14-infected-model mice at two injection time points, 4 h after infection as the early post-infection period and 24 h after infection as the late post-infection period  reported infection from the lung to other organs at 48 h after infection [Based on the results of the in vitro experiments, we conducted in vivo distribution experiments because we considered that the high accumulation contrast of labeled amino acids between n period . When wet 60 min . In the t 60 min . Althougnfection . It can 3H-L-Met and 3H-D-Met except in the brain. Neumann et al. performed bacterial imaging using 11C-L-Met and D-Met in a murine myositis model at 12 h after infection of E. coli strain ATCC 25922 [11C-D-Met compared with 11C-L-Met in imaging at about 60 min after each radiotracer injection. However, their use of a different strain of E. coli than that in the present study (EC-14) might have influenced their results, and the better contrast ratios of 11C-D-Met may have been due to the faster excretion of 3H-D-Met than 3H-L-Met in control mice , which are commonly calculated in tumor imaging, using bacteria ; however, these ratios were 2.11, 2.31, and 2.29; and for each radiotracer, the ratios at 60 min after injection were similar to those at 15 min after injection. In a clinical oncology study, 18F-FDG accumulation in non-small-cell lung cancer showed a more-than-two-fold increase compared with the background [3H-L- and D-Met showed comparable performance in in vivo experiments in EC-14 lung-infection-model mice, and they could be superior to 18F-FDG in terms of detecting bacterial infection at the early stage of infection.At 4 h after infection (early post-infection period), the ratios of accumulation in the infected lung/control lung were 2.80, 2.84, and 1.34 at 15 min after ckground . Thus, w11C-Met imaging, for the reason that our facility is not equipped with a cyclotron or an automatic synthesizer for 11C labeling. However, the results of the whole-body biodistribution are considered equivalent to those of whole-body imaging.As a limitation of this study, we could not perform 11C-L-Met and D-Met, which differ from 3H-L-Met and D-Met only in terms of their radioisotopic labeling, showed comparable performance in in vivo experiments in EC-14 lung-infection-model mice, and are more sensitive than 18F-FDG for detecting bacterial infection at the early stage of infection.