Fucus vesiculosus source  is used as a reducing and capping agent. UV-Vis spectroscopy, XRD, FT-IR, SEM, EDX, and TEM with selected area electron diffraction are used to characterize the synthesized silver nanoparticles (AgNPs). The synthesized AgNPs exhibit a surface plasmon resonance at 430 nm after 24 h. The characterization results showed that AgNPs are crystalline in nature and exhibit mostly spherical shapes with an average diameter of 4-45 nm. Silver nanoparticles showed effective antibacterial activity against representative pathogens of bacteria. The activities of commercial antibiotics were enhanced by impregnation with the synthesized AgNPs. It also shows good fungicidal and anticancer activity against liver and lung cell lines and shows significant antioxidant efficacy (84%) at 10 μg/ml AgNP concentration against DPPH. The utilization of environmentally synthesized AgNPs offers numerous benefits of ecofriendliness and compatibility for biomedical applications.The present research displays the green synthesis of stable silver nanoparticles (AgNPs). The aqueous solution of Fucoidan from  Fucus vesiculosus [Fucus vesiculosus (Fv-fucoidan) as a reducing and capping agent and to explore the potential antimicrobial, antioxidant, and anticancer activities of the synthesized AgNPs.The noble metal nanoparticles have gained great interest in a number of studies due to their potential applications in medical, optical, electronic devices, and water treatment. The most prominent challenge is how to control their sizes and shapes. For this purpose, a large number of reports have been published for the synthesis of metal nanoparticles of diverse structures . The siliculosus –20. Due iculosus , the aimFucus vesiculosus source  and silver nitrate was purchased from Sigma Aldrich, India, and the standard antibiotics and media were purchased from HiMedia laboratories, Mumbai, India. The bacterial strains used in the present study were obtained from Micro Labs, Tamil Nadu, India , and Micro Labs, Chandigarh, India (Bacillus subtilis and Klebsiella planticola).Fucoidan from Fucus vesiculosus source . Briefly, fucoidan-stabilized AgNPs were synthesized by mixing aqueous solutions of fucoidan (10 ml) and silver nitrate  in an Erlenmeyer flask at room temperature. The flask was kept in the orbital shaker at 300 rpm to homogenize the resulting solution for 20 min. The reduction of Ag+ to AgNPs was regularly checked by the UV-visible spectra. The analysis was done using UV-visible spectrophotometer (Perkin Elmer) in the wavelength range 350–650 nm.Colloidal AgNPs were synthesized by reducing the silver nitrate in an aqueous solution of fucoidan from The synthesized AgNPs were purified using deionized water with continuous centrifugation, collected, and dried in hot air oven at 80°C for 2 hours. The dried AgNPs were characterized by various techniques. The crystalline nature was studied by XRD , and the functional groups responsible for reduction of silver ions and stabilization of the formed AgNPs were studied by Fourier transform infrared (FT-IR) spectroscopy (Perkin Elmer). To examine the size and shape of AgNPs, the transmission electron microscope (TEM) (Hitachi S-4500) and scanning electron microscope (SEM) (Philip model CM 200) were used. The elemental analysis was determined by energy-dispersive X-ray spectroscopy (EDS) attached to the SEM –28.Bacillus sp., Serratia pnematodiphila, Streptococcus sp., Klebsiella pneumoniae, Bacillus subtilis, and Klebsiella planticola). The standard antibiotic disks were purchased from HiMedia laboratories . Two different diffusion methods were applied [The antibacterial activity of fucoidan-stabilized AgNPs was examined against six bacterial strains  and Gram-positive  bacteria by adopting the agar well diffusion method [μl) were added to the wells. The AgNP-inoculated plates were incubated for 24 h at 37°C. After that, the inhibition zone around the well was calculated and recorded. The tests were done in triplicates [The antibacterial activity of the synthesized AgNPs were explored against Gram-negative . To determine the mutual effect, each standard antibiotic disks was further impregnated with 25 μl of the freshly prepared AgNPs. The Petri dishes containing 20 ml Mueller-Hinton agar (MHA) were swabbed with 24 h culture of bacterial strains. Standard sterile antibiotic disks are known as positive control, and antibiotics disks impregnated with AgNPs were placed onto the MHA medium inoculated with pathogenic bacterial isolates. The inoculated plates were then incubated at room temperature for 24 h. After the incubation, the zone of inhibition was measured, and the assays were performed in triplicates. The enhancement in the fold area in the zone of inhibition was evaluated by calculating the mean surface area of the inhibition zone generated by an antibiotic (a) and AgNPs impregnated with an antibiotic (b). The fold increase area was calculated by the following equation.a refers to the inhibition zones for antibiotic alone and b refers to the AgNPs impregnated with antibiotic, respectively [Disk diffusion method was used to evaluate the in vitro enhanced antibacterial activity of diverse antibiotics  against the clinical isolates of bacteria  plates. With the help of a sterilized polystyrene tip, about 3 wells (5 mm diameter) were prepared. Diverse concentrations of AgNPs solution  were added to each well on all plates. Then, the plates were incubated at 37°C for 48-78 h. A clear inhibition zone around the wells was detected. For each organism, the inhibition zone diameter was measured (in millimeter).Several pathogenic impacts of fungi have been stated in plants and animals, as well as humans. The opportunistic infections are caused by fungi such as 2. Afterwards, the cells were washed twofold with 100 μl of serum-free medium and starved for 1 h in CO2 incubator. Subsequently, the cells were treated with various concentrations of AgNPs in the range of 1-100 μg/ml and again warmed at 37°C in CO2 incubator. After 24 h incubation, MTT (0.05 mg/ml) was added in every well and again incubated for 4 h. The MTT-containing medium was thrown away and washed with 200 μl phosphate buffer saline solutions. Then, the crystals were dissolved by adding 100 μl of DMSO and mixed well. The appearance of purple blue formazan dye was measured in a microplate reader (570 nm). The cytotoxicity of AgNPs is analyzed using the GraphPad Prism 5 software.The in vitro cytotoxic effect of the synthesized AgNPs on HepG2 and A549 cell lines were evaluated by MTT assay . BrieflyThe formation of free radicals or the deficient removal of reactive oxygen species can develop oxidative damage to biomolecules. These damage leads to numerous sicknesses for human, such as tumor, atherosclerosis, diabetes, maturing, and other degenerative disorders . DPPH , (200), (220), and (311) reflection planes which predicts the face centered cubic structure (fcc) of AgNPs in agreement with the previous study.The crystalline nature of AgNPs was confirmed by XRD . The difThe TEM image of AgNPs showed monodisperse nanoparticles with spherical shape in the size ranges from 4 to 45 nm . MoreoveThe elemental outline of the synthesized AgNPs  showed h−1, showing C-H stretching groups of aromatics, and the low absorption band shows at 2655 and 2325 cm−1, indicating the presence of O-H stretching carboxylic groups. The band at 1560 cm−1 corresponds to amides of N-H bending group, 1363 cm−1 shows aliphatic nitro groups, and 994 cm−1 shows =C-H bending of alkenes groups. The small bands at 702 and 490 cm−1 indicate the presence of alkyl halides.The inorganic biomolecules in the fucoidans responsible for the synthesis of silver nanoparticles were analyzed by using the FT-IR analysis . Silver K. planticola, K. pneumoniae, and Serratia nematodiphila) indicated bigger inhibition zones than the Gram-positive bacteria   .S. nematodiphila (6 mm), penicillin-treated B. subtilis (5 mm), Bacillus sp. (8 mm), and Streptococcus sp. (8 mm), and ampicillin-treated B. subtilis (11 mm) and Streptococcus sp. (9 mm).This might be due to the variety in the composition of their cell walls , 15. TheFusarium sp. (22.43 ± 0.296) > C. albicans (20.07 ± 0.067) > A. niger (17.87 ± 0.241) > A. fumigatus (17.50 ± 0.501) > A. flavus (12.97 ± 0.261). The minimal fungicidal action was noted against A. flavus.The synthesized AgNPs showed excellent fungicidal activity against all chosen clinical isolates Figures  and 10. The liver and lung cancers are the most common cancers causing a lot of death globally. The results of anticancer action imply the high cytotoxic activity of the synthesized AgNPs against the tumor cells (HepG2 and A549) when compared with the standard cyclophosphamide. The cytotoxic effect of fucoidan-mediated silver nanoparticles was higher in A549 cell line than HepG2. As the concentration of AgNPs increases, the cytotoxicity also increases as predicted in μg/ml concentration was recorded to be 84% for DPPH scavenging.The free radical scavenging activity by the DPPH  method showed higher activity in the green synthesized AgNPs when compared with fucoidan and vitamin C (standard). The antioxidant activity of AgNPs displayed significant dosage-dependent inhibition . The maxNoble metals are known to exhibit unique optical properties due to the excitation of SPR. A distinctive surface plasmon resonance (SPR) band was observed at 430 nm after 24 h which is characteristic for AgNPs. As the incubation time was increased, the absorbance of SPR band increased too without band shift. This clearly reflects the development of AgNPs. The stability of the formed AgNPs was evidenced by measuring its UV-Vis absorbance after two months where no alteration in absorbance or shapes of bands was recognized. In the XRD results, the intensity of these peaks 111), (200), (220), and (311) reflects the high degree of crystalline nature of the AgNPs [1, (200),The EDX spectrum additionally shows that the weak signals for oxygen, nitrogen, and carbon were observed. This could be due to the biochemical molecules of Fv-fucoidan responsible for AgNP synthesis and stabilization . In concDespite the fact that it is more pathogenic, it also has more noteworthy harmfulness and produces mycotoxins. Moreover, the increase in AgNPs concentration reduces the antifungal action in a dose-dependent manner . The cytFucus vesiculosus source  at ambient temperature as an effective green reducing and stabilizing agent. The spectroscopic characterization methods display the formation of stable, crystalline, and spherical shape of AgNPs with size ranges from 4 to 45 nm. The synthesized AgNPs show good antimicrobial activity against the selected six pathogenic microorganisms. They also showed a synergistic effect on the antimicrobial activity of the standard antibiotics. Moreover, AgNPs inhibit the cell viability of liver cancer cells lines (HepG2) and lung cancer cell lines (A549). In vitro antioxidant assays illustrated that AgNPs have the potential scavenging activity against DPPH. The conventional chemical methods for synthesizing nanoparticles are costly and time consuming and pose a great threat when disposed in environment, and contact with these chemically synthesized nanoparticles might result in major diseases like skin cancer and lung cancer. Therefore, this green synthesis approach appears to be an ecofriendly alternative to the conventional chemical methods and stands as an effective alternative drug that can be used for biomedical applications in the future.In the current study, we have recognized an ecofriendly, cost-effective, and facile method for the synthesizing AgNPs using fucoidan extract from


Meloidogyne incognita) in okra. The experiments were conducted at research area Department of Nematology in 2017 to 2018 and 2018 to 2019. During this investigation, cruciferous bio-fumigants such as cabbage leaves and cauliflower leaves were used as bio-fumigant sources to protect Okra cv. Hisar Unnat. Fresh and chopped leaf mass of cabbage and cauliflower was incorporated uniformly into a naturally infested field. The initial nematode population in both years was 224 and 256 J2/200 cc soil, respectively. The results of our investigation showed that in both the years okra yield was enhanced significantly by the measures of nematode management. In addition, both of the tested bio-fumigant plants leaves proved to be potentially promising for the management of root-knot nematodes. Among the bio-fumigants, the highest decrease in nematode population, root gall index and increase in yield was observed in cabbage leaves @ 50 t/ha in both years, consecutively.In Haryana, India, only carbofuran is registered for the management of root-knot nematodes. The objective of this study was to investigate the potential of cruciferous bio-fumigants for the management of root-knot nematodes ( Abelmoschus esculentus L. Moench) is a biannual crop with two growing seasons, starting in early March and late July. Based on its rich nutritional contents, this vegetable is very important in India, even though it was originated from the tropics of Afro-Asian countries. It is rich in protein, and the predominant elements found in this vegetable are K, Mg, Na, Ca, and Fe . It also contains vitamins A and B (Meloidogyne spp.) are a hidden threat to okra  released from plant tissues after the decomposition to suppress pests . The terThe experiment was conducted in years 2017 to 2018 and 2018 to 2019 in research area, Department of Nematology, CCSHAU Hisar, Haryana, India , which has subtropical and tropical climate with an annual average temperature of 25.1°C and an average annual rainfall of 459 mm.Abelmoschus esculentus), eggplant (Solanum melongena), and tomato (Solanum lycopersicum) for seven years.Root-knot nematode inoculum was obtained from okra infested field of Hisar, Haryana, India. It was maintained and cultured on tomato variety ‘Hisar arun’ and species identified by using the perineal pattern . The fieThe plants were observed regularly, and watering, hoeing, etc., were done whenever required. Fertilizers were applied according to the recommended dosage for okra.Brassica oleracea var. botrytis) and cabbage (Brassica oleracea var. capitata), was evaluated over two years. Plot size was 3.0 × 2.1 m. The initial nematode population (INP) in the soil before the incorporation of leaves was 224 J2/200 cc and 256 J2/200 cc in 2017 to 2018 and 2018 to 2019, respectively. Fresh and chopped leaf mass of cruciferous bio-fumigants was incorporated uniformly into infested field, 15 days before sowing. After 15 days of incorporation of leaves, nematode populations were counted from treated plots . The percentage increase and decrease in the yield over the control were calculated by the following formula:M. incognita juveniles (J2) were counted at 40 × magnification.Roots from these plants were indexed for galling and egg mass presence on a scale from 1 to 5 where 1.0 = no galls or egg masses, 2.0 = 1-10 galls or egg masses, 3.0 = 11-30, 4.0 = 31-100 galls or egg masses, and 5.0 = more than 100 galls or egg masses . For thewww.hau.ernet.in). The comparisons in treatments were made by critical difference at the 5% level of significance. Necessary transformations of data were done where applicable. The relationships between the number of galls and the yield were determined using regression analysis with Excel 2016.The data were subjected to RBD using OPSTAT program available online at CCSHAU Hisar University website  followed by cabbage leaves @ 50 t/ha (215 J2/200 cc soil). The regression studies showed (r² = 0.0651).The multiplication of  results  indicates showed  negative2/200 cc soil) followed by cauliflower leaves @ 50 t/ha (181 J2/200 cc soil). A highest reduction in the gall index of okra roots was obtained from field plots incorporated with cruciferous bio-fumigants but not in untreated check (r² = 0.0591).After the incorporation of cruciferous bio-fumigants, the highest decrease in nematode juvenile population was observed in cabbage leaves @ 50 t/ha (151 Jed check . The cared check . The fined check  negativeM. incognita in okra. Our findings are in agreement with Meloidogyne javanica in pepper. As reported by many scientists, field experiments in which Brassica juncea and Brassica rapa were grown as green manure crops resulted in 91 to 95% mortality of encysted eggs of Globodera pallida (In recent years, bio-fumigation has emerged as an effective non-chemical alternative to manage nematode pests. However, these results aim to be only indicative of the potential use of cruciferous bio-fumigants for nematode management and need to be validated by future trials in farmers field conditions, as well as different combinations of bio-agents with cruciferous bio-fumigants should also be tested to verify a potential synergism among different practices. The experimental study indicated that cruciferous bio-fumigants have provided a satisfactory nematode suppression, confirming previous findings of some researchers . In cruc pallida . On the  pallida , and theMost used nematicides banned from world markets and trends toward natural farming and sustainable agriculture continue to escalate; more research and outreach effort are necessitated towards developing and disseminating information on alternative PPN management strategies. One such strategy is the use of cruciferous bio-fumigants against root-knot nematode. Although a plethora of research have been conducted on using these crops for root-knot nematode management, agricultural stakeholders remain to be convinced to integrate bio-fumigation into their INM practices. Indeed, it is unlikely that bio-fumigation as a standalone technique will eliminate target PPNs in soil, but this technique can easily be integrated with other strategies such as soil solarization, minimal use of nematicides, use of resistant varieties, bio-control agents, etc., in a cost-effective manner to provide acceptable levels of nematode management.


LAMP on-site sweetpotato virus detection workflow. •LAMP assays were developed to detect the three main sweetpotato viruses.•Unpurified extracts could be used, and results were obtained from 5 to 45 minutes.•The sensitivity was higher than RT-PCR and PCR assays.•The assays showed 100 % concordance with standard virus indexing in laboratory.•On-site, in-field testing at different location in Kenya demonstrated high accuracy. Globally, Sweet potato feathery mottle virus (SPFMV) and Sweet potato chlorotic stunt virus (SPCSV) occur frequently and in combination cause sweetpotato virus disease (SPVD). Many viral diseases are economically important and negatively impact the production and movement of germplasm across regions. Rapid detection of viruses is critical for effective control. Detection and quantification of viruses directly from sweetpotato remains a challenge. Current diagnostic tests are not sensitive enough to reliably detect viruses directly from the plant or require expensive laboratory equipment and expertise to perform. We developed a simple and rapid loop‐mediated isothermal amplification (LAMP) assay for the detection of SPFMV, SPCSV and begomoviruses related to sweet potato leaf curl virus (SPLCV). Laboratory validation recorded 100 % diagnostic sensitivity for all the three viruses. The LAMP assays were customized for field testing using a lyophilized thermostable isothermal master mix in a ready-to-use form that required no cold chain. The average time to positivity (TTP) was: SPFMV 5−30 min, SPCSV 15–43 min s and begomoviruses 28−45 mins. LAMP on-site testing results were comparable to PCR and RT-PCR confirmatory laboratory tests. The LAMP assay is a powerful tool for rapid sweetpotato virus detection at a reasonable cost and thus could serve as quality control systems for planting materials. However, several other viruses (mostly potyviruses) are also known to cause synergistic diseases with SPCSV   and the tato sap . Howevertato sap ,b. The ktato sap .Challenges in the detection of sweetpotato viruses have been reported to be caused by low viral titers  the occuThe loop-mediated isothermal amplification (LAMP) method, developed by LAMP is gaining popularity in plant health diagnostics because of its time efficiency and cost effectiveness. The LAMP assay has been successfully used to detect plant pathogens; phytoplasma infecting papaya, potato, coconut, periwinkle, and some insect hosts . The cur22.1I. setosa  from the International Potato Centre research support unit, Peru (CIP-RSU Lima) and (n = 10) from the Kenya Plant Health Inspectorate Service, Plant Quarantine and Biosecurity Station, Kenya (KEPHIS-PQBS). The virus/es present in each sample are shown in . setosa  combined. setosa ,b. AntisSections  and 2.4.2.2Three leaves  of a test plant were sampled into 4"x6", 150 microns plastic extraction bags. Three one cm sweetpotato discs (one from each sampled leaf) were sampled in duplicate from the same leaf tissue to compare two parallel nucleic acid extraction methods. Total nucleic acid was extracted from one of the duplicates using the Ambion Kit , as described by the manufacturer. The kit-extracted RNA/DNA purity and concentration were checked using a NanoDrop ND-1000 Spectrophotometer (Thermo Fischer Scientific) followed by a quality check on 2% agarose gel. RNA/DNA concentrations were standardized to 100 ng/μL before use in amplification assays. For LAMP, we also used the alkaline polyethylene glycol (APEG) quick extraction method  with sli2.3RNA viruses (SPCSV and SPFMV) were detected using a SuperScript III One-Step RT-PCR with Platinum Taq Kit , using the primers described by . A 20 μl2.4Reverse transcription Quantitative PCR assays (RT-qPCR) for RNA viruses were performed as described by . The assQuantitative PCR assays (qPCR) for the DNA virus SPLCV was done as described by Ling et al. (2010). A PCR Master Mix Reagents kit (Applied Biosystems) was used. Briefly, 1 μl of template DNA, 0.4 μM each of forward and reverse primer, 0.2 μM TaqMan Tamra probe, 12.5 μl of the 2× Master Mix (Applied Biosystems), and 10.15 μl nuclease free water were mixed in a 25 μl reaction volume. The following real-time PCR thermal cycler conditions were used: 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 1 min and extension at 72 °C for 1 min. To account for pipetting differences, each sample was run in triplicate on each plate and their threshold cycle (Ct) values averaged during data analysis. In addition, non-template water controls (NTC) as well as positive  were included. Real-time PCR reactions were performed on Quant-5 studio (Applied Biosystems).2.5http://www.ebi.ac.uk/Tools/msa/clustalo/) to obtain the consensus sequences. Due to diversity of Sweepoviruses, LAMP primers were modified by incorporating degenerate bases wherever necessary. Primers  that recognize six distinct regions of the viral coat protein were designed. The sequences of the primer sets are given in LAMP Designer software – OptiGene  was used to design the primers. LAMP primer parameters were followed as described by . To desiA full factorial design experiment was adopted to expedite the optimization process as multiple input factors were manipulated in determining their effect on a desired output. A series of reactions were performed with the SPFMV, SPCSV and SPLCV primers to obtain optimal conditions. Key considerations in the development and optimization of LAMP assays were: effect of extraction method (Ambion kit versus APEG quick extraction), type of tissue to be analyzed , purity of FIP and BIP , specificity of primers, optimal LAMP reaction temperature  and LAMP reagents  were performed. All LAMP assays were run on a rechargeable, portable Genie® II , at 65 °C for 30 min. A positive reaction was signified by an exponential increase in fluorescence (δR). Peak fluorescence ratio on the amplification rate curve, with a threshold value of 0.02 indicated the time to positivity (Tp). Specificity of sweetpotato virus amplicons was determined after a melt curve (Tm) analysis. This was achieved by heating RT‐LAMP products to 98 °C for 1 min, then cooling to 80 °C and decreasing at 0.05 °C/s. Melting curve analysis was used to distinguish between true and false positive reactions. Similarly, RT-qPCR was conducted for SPCSV and SPFMV, while qPCR for SPLCV was performed as described in Section To assess the effect of extraction method on LAMP assay sensitivity, a serial dilution of 102.64, dNTPs, and a ds-DNA binding dye (FAM detection channel). On the other hand, for ‘dry’ LAMP the freeze‐dried isothermal master mix ISO-DR004-RT  was used and is similar to ISO-DR002-RT in its final composition but contains undisclosed proprietary reagents. Each reaction was resuspended to 25 μl and LAMP reaction mix comprised 15 μl of resuspension solution, 5 μl of 10x concentrated primer mix, corresponding to the virus to be tested , 4 μl NFW and 1 μl of RNA/DNA template added into individual reaction mix. The ‘wet’ and ‘dry’ assay were both used for lab validation. Lyophilized ‘dry’ LAMP assay was used for on-site detection of SPFMV, SPCSV and SPLCV. LAMP assays were run as described in 2.5.The 25 μl ‘wet’ LAMP reaction mix comprised of 15 μl of isothermal master mix ISO-DR002 , 5 μl of 10X the primer set to be tested , 4 μl NFW and 1 μl of RNA/DNA template. The 10X primer combination contained 2.0 μM each of forward and reverse inner primers (FIP and BIP respectively), 0.5 μM each of forward and reverse outer primers , and 1.0 μM each of forward and reverse loop primers . The isothermal master mix ISO-DR002-RT  contains a proprietary fast novel DNA polymerase, proprietary thermostable inorganic pyrophosphatase, optimized reaction buffer, MgSO2.7Operational performance of ‘dry’ LAMP assays for the detection of SPFMV, SPCSV and SPLCV, respectively, were evaluated on-site in four different geographical conditions in Kenya. These were in the diverse Central, Kakamega, Eastern and Coast regions. Operating temperature average daily max °C (range of daily max) at the time of on-site detection and altitude are given in 2.8t-test procedure in SAS  was performed to compare the differences of extraction methods on LAMP assay results. Diagnostics sensitivity and specificity of LAMP assays were calculated. Sensitivity is the proportion of true positives that are correctly identified by a test, whereas the specificity is the proportion of true negatives that are correctly identified by the test. Sensitivity (SE), specificity (SP) and accuracy (AC), positive and negative predictive values  and the kappa index of concordance (k) were calculated as previously described by  . Specificity of the SPFMV primers was evaluated against a panel of other potyviruses; Sweet potato virus G (SPVG), Sweet potato virus C (SPVC), Sweet potato virus 2 (SPV2)  . The SPFm values for specific products for COX, SPFMV, SPCSV, and SPLCV were found to be 84.7 °C ± 0.4, 85.7 °C ± 0.5, 85.6 °C ± 0.45, and 88.7 °C ± 0.4 °C, respectively : COX 10–28, SPFMV 5−30 min., SPCSV 15–43 min s and sweepoviruses 28−45 mins A. To conectively B. Examplively , a total of 96 samples were tested in the field by ‘dry’ LAMP assay for SPFMV, SPCSV and SPLCV and compared to RT-PCR/PCR results of the same leaves in the lab. The results are detailed in 4I. setosa combined with ELISA and RT-PCR/PCR under laboratory conditions. This report constitutes the first validation using diagnostic parameters in the development of field adaptable LAMP assays for SPFMV, SPCSV and SPLCV. Rapid detection is important for disease management in sweetpotato seed systems. LAMP assays were developed in this study for the detection of SPFMV (and related potyviruses), SPCSV and SPLCV (and related begomoviruses), which are the most common viruses infecting sweetpotato in Kenya and sub-saharan Africa, and their operability further evaluated under field conditions in Kenya. Our results show a perfect agreement Cohen’s kappa index of 1 between our LAMP assays for these viruses and the gold standard of grafting to Key to the adoption of field testing is the availability of a quick extraction method that negates the need of RNA/DNA purification that is difficult to perform under field conditions. APEG quick extraction has previously been used on blood, insect and plant tissue . Howson Another aspect that can limit on-site LAMP detection assays are the use of ‘wet’ LAMP reagents as they require utilization of cold storage to protect the integrity and stability of reagents . We utilThe LAMP assays we developed detected all the three viruses with a sensitivity of 100 % as compared to standard virus indexing with a set of 100 plants  at MugugI. setosa, leading to virus symptom expression . We focused our validation on testing of leaf tissues, since that would be the most likely testing approach on a continent where vines are the predominant multiplication material. However, while in the field, we also tested ten root samples from individual plants and roots gave a comparatively fast TTP and results reproduced those obtained with leaf tissue of the same plant . Roots are occasionally used as propagation material in in drought-prone areas and are good reservoirs for viruses . Pieces Despite that LAMP assays for detection of SPCSV and SPLCV were specific when considering positive amplification curves, some unexpected observations were witnessed for these assays. The SPCSV assay produced a ‘pseudo anneal derivative’ in a melting curve analysis when the reactions were negative for amplification. Although this is not a large issue since any lack of an amplification curve would lead to consider the reaction negative, it could potentially lead to confusion. This was resolved by the design of new primers, which did not produce the pseudo anneal derivative, and additionally led to more rapid amplification ; Table 2The operational field performance of our LAMP assays was evaluated in four different geographical sweetpotato growing regions and results compared to RT-PCR and PCR in the lab, using desiccated leftovers of the same leaves tested in the field . ResultaPhytophthora spp. costs from ∼USD 7 per test and significantly cheaper than laboratory testing  developed a LAMP assay recipe for the detection of Ralstonia from different samples  that costs approximately USD 3 per sample. This is evident that LAMP assays can be customized to reduce cost. One of the setbacks of the use of molecular methods is the increased costs compared to the traditional methods. We envisage that LAMP will reduce the time and cost. The current molecular test for sweetpotato viruses costs USD 30 per sample per virus at KEPHIS. Virus indexing and testing for 10 sweetpotato viruses at CIP Lima costs USD 120, which translates to USD 12 per sample and virus tested. Based on costing key reagents, we estimate LAMP to cost USD 6 per sample per virus. Comparatively, the LFD kits for detecting  testing . Wang anIn conclusion, our LAMP assays have the potential to reliably and accurately detect SPFMV, SPCSV and SPLCV. Further, the assay utilizes simple and relatively inexpensive equipment, which renders it promising for use in resource-poor settings. Indeed, we have been able to adapt a kit with single use plastic 1 mL pipettes and 1 μL inoculation loops to simplify the procedure even further without loss of fidelity. We propose this LAMP assay can be used for: field surveys, monitoring of the phytosanitary status of pre-basic seed production in quarantine, or certification program. This can support the production of pathogen-free plant material entering the seed system. However, key to adoption in sub-Saharan Africa will be adequate access to affordable kits. The simplicity and robustness of these kits will make them suitable for the rapid assessment of sweetpotato viruses by plant health inspectors.Bramwel W. Wanjala: Data curation, Formal analysis, Investigation, Methodology, Writing - original draft, Writing - review & editing. Elijah M. Ateka: Investigation, Methodology, Validation, Writing - review & editing. Douglas W. Miano: Investigation, Methodology, Validation, Writing - review & editing. Segundo Fuentes: Validation, Writing - review & editing. Ana Perez: Validation. Jan W. Low: Conceptualization, Writing - review & editing. Jan F. Kreuze: Conceptualization, Investigation, Methodology, Validation, Writing - review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Subclinical bacterial infections (biofilms) are strongly implicated in breast augmentation failure due to capsular contracture, and while these infections are generally ascribed to common skin commensals, this remains largely unsubstantiated through robust cultivation independent analyses. To determine capsule biofilm microbial community compositions, we employed amplicon sequencing of the 16S rRNA gene using DNA extracted from breast implant capsule samples. These cultivation independent analyses revealed that capsule associated biofilms are more diverse than canonical single-species infections, but have relatively low diversity (~ <100 species) compared to many host-associated microbial communities. In addition to taxa commonly associated with capsular contracture, the biofilms analyzed comprised a number of taxa that escaped detection in cultivation-dependent work. We have also isolated several key taxa identified through the culture-independent analyses. Together our analyses reveal that capsule biofilms are more diverse than cultivation studies suggest and can be heterogeneous within an individual capsule, between breasts of the same patient, across similar implant types, and over a range in severity of contracture. The complex nature of these communities requires further study across a broader suite of patients in addition to higher resolution analyses including metagenomics to better assess the fundamental role of microorganisms in capsular contracture. The human body is comprised of more than one microbial cell for every human cell  and thesIllness related to microbe-host interactions (dysbiosis) is best thought of as a disruption to the composition and activity of the healthy microbiome. Surgical procedures, including implantation, often cause dysbiosis as a result of immune responses, introduced microbial community members, or the alteration of the physical-chemical properties of the tissue environment. Colonization of surgical implants by microorganisms, both innate and foreign, and the subsequent development of microbial biofilms on implant surfaces often leads to complications including inflammation and other immune responses, implant failure, or disruption of optimal wound healing and scar tissue formation . Prevent, and these are among the most widely performed surgical implantations. Subclinical bacterial infections (biofilms) are strongly implicated in capsular contracture [All implanted foreign bodies induce a host reaction consisting of production of a capsular layer that surrounds the foreign material. In the case of breast implants, the capsule can vary from soft and pliable (Baker grade I) to a pathologic contracted layer causing the implant to become distorted, hard, and painful (Baker grade IV) , 18–20. tracture , 22, andtracture –25. Whiltracture . New pretracture , 28.Staphylococcus epidermidis has been implicated in biofilm formation on implant capsules based on its recovery in standard culture dependent medical studies [Staphylococcus taxa in newer culture-independent studies [S. epidermidis is classically considered non-pathogenic, though it is increasingly associated with sub-clinical infections, in particular those involving prosthetic devices [Staphylococcus sp. almost certainly play a role in the formation of capsular biofilms [ studies , as well studies , 31. S.  devices –34. Whilbiofilms , 36, thebiofilms , 31. Det5 to 5 x 107 copies g-1 of tissue extracted , B is the read count for a given sequence in the blank, and Bmax is the read count of the most abundant sequence in the blank. On average this blank correction decreased the number of observed sequences by 4%, and at a maximum, it decreased the number of observed sequences by 16%. We thus refer to this blank corrected data throughout the rest of the paper.Amplicon sequencing of 16S rRNA gene copies from low biomass samples can also be prone to contamination associated with library preparation. To evaluate such contamination in our data we sequenced both our extraction and PCR blanks, which mostly yielded sequences affiliated to the Proteobacteria, Firmicutes, and Actinobacteria phyla. To correct microbial community profiles for such possible contamination, we proportionally removed reads from samples based on the relative abundances of sequences recovered from our extraction blanks. The most abundant sequence in the blank was used to estimate the fraction of reads in a sample that could have resulted from contamination, conservatively assuming all sample reads with that sequence were derived from contamination. We then subtracted reads from other sequences based on their proportional abundance in the blank. This blank correction can be written as:Rhodococcus species (sp.) (range 99.8–99.9%), while capsule C7L was largely comprised of Firmicutes of the unclassified Bacillales sp. (range 99.7–99.8%) (Rhodococcus sp. (13.2–99.6%), Enhydrobacter sp. (up to 77.6% in a single replicate), and Finegoldia sp. (up to 85.8% in a single replicate) across 6 separate replicates (Escherichia-shigella sp. (0–99.8%), Rhodococcus sp. (0–99.1%), unclassified Burkholderiaceae sp. (0–99.6%), Staphylococcus sp. (41.0%) and Acinetobacter sp. (0–18.7%)  analyses of microbial community composition were conducted on 4 contracted capsules, with a range in bacterial loads, and the degree of heterogeneity within a given contracted capsule was variable . Of the 7–99.8%) , across 7–99.8%) . Similarplicates . In caps0–18.7%) . Notablypies g-1 .Staphylococcus sp. (range 0–99% present in 15/24 samples), Rhodococcus sp. (range 0–83% present 15/24 samples), unclassified Bacillales sp. , Escherichia-shigella sp. , Pseudomonas sp. , and Cutibacterium sp.   . The othRhodococcus sp., Bacillus sp., Staphylococcus sp., and Micrococcus sp.  and are similar in diversity to healthy breast tissue . Organisms of the same species are well known to exhibit differences in genomic composition and metabolic potential— strains . Notablytracture , 35, 36.crobiome , but alscrobiome , 56. TheWe evaluated relationships between implant type, plane of placement, Baker contracture grade, and microbial community composition through hierarchical clustering analyses . In hierOur analyses reveal that microbial communities associated with pathologic and non-pathologic contracted capsules are considerably more diverse than previously appreciated. Taxa from 4 main phyla- the Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes- are most commonly present and at appreciable relative abundances. This observation is consistent across multiple other cultivation dependent and cultivation independent studies. By contrast, however, we also find a number of other taxa that are generally present at variable and low relative abundances that sometimes can represent appreciable fractions of the community. This result appears to contrast with previous studies, but reveals that methodological variability across studies may lead to differing results and conclusions. This highlights a need for the cosmetic surgery community to develop standards of best practice if amplicon sequencing approaches are to be adopted in research and clinical practice, more broadly. In particular, these standards should: 1) address contamination; 2) select regions of the 16S rRNA or other genes to be used; 3) optimize DNA extraction and amplification protocols; 4) establish minimum information standards for metadata; and 5) develop consensus on sequencing platforms and depth. Our findings, that capsule associated microbial communities are diverse and highly variable, suggests that mitigation of microbial capsule colonization could be best addressed through personized approaches that consider the broader patient microbiome. As noted above, however, standards of best practice across the community could promote further research, by multiple groups, that would be needed to dramatically increase the number of observations and provide the statistical power needed to establish cause and effect relationships.Ethics approval for this study was obtained through the University of British Columbia Clinical Research Ethics Board under “Biofilm Study” H16-01002.Capsule samples analysed in this study were all collected in the private practice of author NJC from patients that underwent revision surgery consisting of implant exchange or removal. The sample series represents 17 consecutive cases between February of 2015 and June of 2016. All capsule specimens were collected from aesthetic patients except capsule 1, which was collected from a reconstruction surgery. Given that a goal of this study was to examine biofilm composition across a range of pathological and non-pathological capsules, we analysed a suite of samples that varied from Baker grade I to IV. Reasons for surgery included implant malposition, implant rupture, pathological capsular contracture, or patient desire to change implant type. Capsular tissue samples were removed aseptically from the patient and placed separately in sterile containers, containing saline. In most cases one sample was taken from a visually representative area of the capsule of each implant. In one case, C14, an additional sample was taken from one of the implant capsules because of the presence of the distinctly different appearance in areas of the capsule surrounding the implant. The implants were processed for microbiological analysis within approximately 24 hours of removal.∘C for 30 minutes. 1 μl of lysed sample was used in the PCR with the universal primers 27F/1492R, to amplify full-length bacterial 16SrRNA genes. PCR amplifications were carried out with the following cycling conditions; initial denaturing at 95 ∘C for 3 min, 30 cycles of 95 ∘C for 30 s, 48 ∘C for 30 s, and 72 ∘C for 60 s, followed by a final elongation step at 72 ∘C for 10 min. PCR products were Sanger sequenced with forward and reverse primers (27F/1492R) by GeneWiz LLC.Aseptically cut tissue sections were swabbed for culturing onto LB agar. Plates were incubated at 37°C for 1–7 days. Pure colonies selected for sequencing were grown up overnight in LB broth 37°C, then lysed at 95 ® Soil DNA Isolation Kit, as per manufacturer’s instructions. Resulting DNA was stored at -20 ∘C. The quality and quantity of genomic DNA were measured on a NanoDrop® ND-1000 spectrophotometer (Thermo Scientific) and by PicoGreen .Microbial DNA was extracted from approximately 0.25 g of breast capsular tissue using the Mobio PowerMax∘C for 2 min, followed by 30 cycles of denaturation (95 ∘C for 20 s), annealing (55 ∘C for 15 s), and elongation (72 ∘C for 5 min), with a final elongation step at 72 ∘C for 10 min. A nested-PCR approach, using primers 27F and 1492R for the initial amplification, followed by a second amplification using primer pair 515F/806R under the conditions outlined above, was adopted for samples in which the original PCR did not yield any products. Equimolar concentrations of prepared amplicon samples were pooled into a single library by using the Invitrogen SequalPrep kit. The amplicon library was analysed on an Agilent Bioanalyzer using the High Sensitivity dsDNA assay to determine approximate library fragment size, and to verify library integrity. Pooled library concentration was determined using the KAPA Library Quantification Kit for Illumina. Library pools were diluted to 4 nM and denatured into single strands using fresh 0.2 N NaOH as recommended by Illumina. The final library was loaded at a concentration of 8 pM, with an additional PhiX spike-in of 5–20%. Sequencing was conducted at the University of British Columbia Sequencing and Bioinformatics Consortium (http://sequencing.ubc.ca) All raw sequence data (including blanks) were submitted to the SRA database under accession PRJNA632935.Bacterial and archaeal 16S rRNA gene fragments from the extracted genomic DNA were amplified using primers 515F and 806R. Sample preparation for amplicon sequencing was performed as described as . In brie5′-AGAGTTTGATCCTGGCTCAG) and DW519R (5′-GNTT TACCGCGGCKGCTG). Each amplification reaction (20 μL) contained PCR certified water (4μL), SsoFast™ EvaGreen master-mix (10 μL), and 3 μM primer (2 μL). Amplification of the 16S rRNA gene was performed with an initial denaturation step , followed by 45 cycles of: denaturation ; annealing ; and elongation . This was followed by a melt curve analysis for assessing amplicon specificity . Bacterial DNA standards, ranging from 102−108, non-template controls, and sample DNA were all run in duplicate. The qPCR assays were tested for inhibition by adding known copies of E. Coli 16S rRNA genes and comparing the resulting measured copy numbers to the expected copy numbers.Bacterial DNA was quantified by quantitative polymerase-chain reaction (qPCR) using the SsoFast™ EvaGreen assay (Bio-Rad™) and a CFX96 Real-Time Detection System (Bio-Rad™). The 16S rRNA gene was targeted using bacterial-specific primers, 27F, . Sequences were chimera checked using VSEARCH [http://www.mothur.org/wiki/Silva_reference_files). All data was visualized in RStudio  [Sequences were processed using Mothur and the Miseq protocol . Sequenc VSEARCH  and readm, 2015) . For genhttp://www.genecodes.com). 16S rRNA gene sequences were compared to available databases using the Basic Local Alignment Search Tool (BLAST) [Full-length 16S rRNA sequences were quality checked using the Sequencher software package  . To dete (BLAST)  then imp (BLAST) . All sub (BLAST) . Maximum


However, a significant amount of patients show no response to immunotherapy, while issues on its reliable response interpretation exist. Aim of this study was to investigate the phenomenon of early disease progression in 2-deoxy-2-(18F-FDG PET/CT were enrolled. All patients exhibited progressive metabolic disease (PMD) after two ICIs’ cycles according to the European Organization for Research and Treatment of Cancer (EORTC) criteria, and were characterized as unconfirmed PMD (uPMD). They were further followed with at least one PET/CT for either confirmation of PMD (cPMD) or demonstration of pseudoprogression remission. Patients were also evaluated with the PET Response Evaluation Criteria for Immunotherapy (PERCIMT). Moreover, in an attempt to investigate immune activation, the spleen to liver ratios  of 18F-FDG uptake were measured.Thirty-one patients under ICIs serially monitored with n = 26) had a median OS of 10.9 months [8.5–NA], while those with pseudoprogression (n = 5) did not reach a median OS [40.9–NA]. Respectively, after application of PERCIMT, 2/5 patients of the pseudoprogression group were correctly classified as non-PMD, reducing the uPMD cohort to 29 patients; eventually, 26/29 patients demonstrated cPMD (89.7%) and 3/29 pseudoprogression (10.3%). One further patient with pseudoprogression exhibited transient, sarcoid-like, mediastinal/hilar lymphadenopathy, a known immune-related adverse event (irAE). Finally, patients eventually showing cPMD exhibited a significantly higher SLRmean than those showing pseudoprogression after two ICIs’ cycles (p = 0.038).Median follow up was 69.7 months [64.6–NA]. According to EORTC, 26/31 patients with uPMD eventually showed cPMD (83.9%) and 5/31 patients showed pseudoprogression (16.1%). Patients with cPMD (PET/CT, performed already after administration of two ICIs’ cycles, can identify the majority of non-responders in melanoma immunotherapy. In order to tackle however, the non-negligible phenomenon of pseudoprogression, another follow-up PET/CT, the usage of novel response criteria and vigilance over emergence of radiological irAEs are recommended. Moreover, the investigation of spleen glucose metabolism may offer further prognostic information in melanoma patients under ICIs. In recent years the clinical application of novel immune checkpoint inhibitors (ICIs) has constituted a major breakthrough in the management of advanced melanoma, leading to unprecedented response and survival rates , 2. ICIsImmunotherapeutic agents act markedly different than usual cytotoxic approaches, notably by generating inflammations rather than direct lysis. This unique mechanism of action can lead to novel response patterns, which pose relevant challenges in the interpretation of treatment response by conventional imaging approaches . Pseudop18F)fluoro-D-glucose (18F-FDG) positron emission tomography/computed tomography (PET/CT), leading to the proposal of the respective, metabolic criteria, namely the PET/CT Criteria for Early Prediction of Response to Immune Checkpoint Inhibitor Therapy (PECRIT) , 2217, 2mean, SUVmax) of the liver and the spleen, if without disseminated metastatic disease, were measured in the PET/CT scans performed before treatment and after the first 2 cycles of ICIs. SUV values of the spleen were calculated from a central volume of interest (VOI) on the spleen, while the respective values of the liver were calculated from a VOI placed on the right liver lobe  estimated. For univariate comparison of OS, a log-rank test was used. Statistical analysis was performed using R version 4.0.2  and R packages survival and prodlim. The results were considered significant for n = 31 patients) eventually demonstrated cPMD (83.9%), while 5 of them showed pseudoprogression (16.1%). Concerning the pseudoprogression group, 4/5 patients were under ipilimumab, while 1/5 patient was under combination treatment of ipilimumab/nivolumab. The median time between the emergence of signs of uPMD and confirmation of PMD was 1.5 months [1.4–1.9 months], while the median time between uPMD and remission of pseudoprogression was 2.3 months [1.5–NA months] (p = 0.092). The demographic characteristics, imaging findings and survival data of the five patients with pseudoprogression according to EORTC are summarized in Table Each patient underwent a median number of 3 PET/CT scans  between start of treatment and clinical disease progression. According to EORTC, 26 patients of the initial uPMD cohort .Compared to EORTC, the application of PERCIMT classified 2/5 patients of the above-mentioned pseudoprogression group as stable metabolic disease (SMD), since they did not fulfill the requirements for progression according to these criteria. In particular, one patient developed two small, cervical, With further consideration to the pseudoprogression group, one of the remaining three patients developed a transient, almost symmetric, sarcoid-like involvement of the mediastinal and hilar nodes, which is recognized as a possible, special type of irAE , accompamean of patients eventually showing cPMD was 0.91 (median = 0.92), while the mean SLRmean of those eventually showing pseudoprogression was 0.85 (median = 0.85) (p = 0.062). Respectively, the baseline mean SLRmax of patients eventually showing cPMD was 0.85 (median = 0.87), compared to a mean SLRmax of 0.89 (median = 0.85) for patients with pseudoprogression (p = 0.625).After performing semi-quantitative (SUV) measurements in the spleen and liver, the following SLR results at baseline (before commencement of immunotherapy) imaging were revealed: the mean SLRmean of 0.96 (median = 0.97), while patients with pseudoprogression had a mean SLRmean of 0.88 (median = 0.88) (p = 0.038). Finally, the mean SLRmax values for patients with cPMD and pseudoprogression were 0.95 (median = 0.92) and 0.84 (median = 0.80), respectively (p = 0.112).With regard to the measurements derived from PET/CT after the first 2 cycles of ICIs, patients eventually showing cPMD had a mean SLRn = 26) had a median OS of 10.9 months [8.5–NA], while those with pseudoprogression (n = 5) did not reach a median OS [40.9–NA] (p = 0.108) , we refrained from performing the respective OS comparison based on PERCIMT. Figure Median follow up of the patient cohort from start of treatment was 69.7 months 64.6–NA]. According to EORTC, patients showing cPMD (08) Fig. . Due to .6–NA. Ac18F-FDG PET/CT in oncology [18F-FDG PET/CT, inevitably, raising the issue of specificity.Through the restoration of T cell function, ICIs activate the adaptive immune system to produce enhanced anti-tumor responses . The actoncology . In thisoncology —leads to18F-FDG PET/CT under ICIs. Following the recommendations of the novel response criteria to immunotherapy [The reliable differentiation of true disease progression from pseudoprogression is clinically relevant, since it can identify non-responders who will have a shorter duration of benefit from the treatment , 33. Howotherapy , 18, we mean after the first 2 cycles of treatment than those showing pseudoprogression.There are three major findings from our analysis. Firstly, the majority (83.9%) of patients with signs of early metabolic progression on PET/CT—already after administration of two ICIs cycles—eventually show confirmed progressive disease. On the other hand, approximately every sixth patient with initial signs of metabolic progression has eventually pseudoprogression. Importantly, survival analysis revealed a longer OS for patients with pseudoprogression compared to those with cPMD; this difference in survival may not be statistically significant, which is very likely attributed to the small number of patients in the pseudoprogression group, but a clear trend was recognized in the respective Kaplan–Meier curves. Secondly, we demonstrate that the incidence of pseudoprogression can be markedly decreased after application of novel response criteria and identification of signs of irAEs. Thirdly, patients eventually responding to ICIs with cPMD exhibit a higher SLRThe detection of non-responders already after administration of the first ICIs cycles carries significance, since it can limit the treatment-associated toxicity and financial burden in patients that are unlikely to profit from ICIs . In our On the other hand, the identification of uPMD at an early time point comes at a cost: the misdiagnosis due to the phenomenon of pseudoprogression of some late responders as PMD would deprive these patients of the beneficial effect of immunotherapy. Indeed, a non-negligible number of patients (16.1%) eventually showed a subsequent remission of the initial uPMD. This finding suggests that pseudoprogression is not uncommon, which is in line with the results of a recent study by Pires da Silva et al. In that study, involving 140 melanoma patients treated with combined immunotherapy, one-third of all patients with progressive disease -according to RECIST 1.1—had eventually pseudoprogression and exhibited similar survival compared with non-progressors .18F-FDG-avid lesions is more predictive of clinical outcome than SUV changes during melanoma immunotherapy [These challenging results call for a more detailed analysis of the specific PET/CT findings of the 5 patients with pseudoprogression, in order to possibly identify imaging characteristics suggestive of this phenomenon and develop approaches that could address it. Specifically, in 2 patients of the pseudoprogression group—both of which showed a very good clinical response—misdiagnosis could be tackled after application of the recently proposed PERCIMT, instead of the EORTC criteria. The cornerstone of PERCIMT is the finding that the absolute number of newly emerged otherapy . In partotherapy . The hitotherapy , 38–41.18F-FDG in up to 5 measurable tumor/target lesions corrected for lean body mass (SULpeak), as suggested by PERCIST. However, imPERCIST have two major differences in comparison with PERCIST: firstly, PMD is not defined by the appearance of new lesions but exclusively by the increase of SULpeak. Secondly, the selection of target lesions at follow-up scan(s) is based on the 5 hottest lesions among all lesions in each scan—including newly emerging lesions -, irrespective of the distribution of lesions at baseline PET/CT imaging [Besides PERCIMT, various novel metabolic response criteria have been recently proposed as alternative approaches to the conventional PET criteria—mainly the PET Response Criteria in Solid Tumors (PERCIST)—which were based on data derived from cohorts under cytotoxic therapies . These n imaging . IndicatWith further consideration to the pseudoprogression group, one patient developed a transient sarcoid-like lymphadenopathy of the mediastinal and hilar nodes. He eventually showed a very good clinical response. In clinical practice, the emergence of this response pattern during immunotherapy can create a diagnostic dilemma for imaging specialists and clinicians, since it may mimic disease progression. Based on the present findings and the steadily growing literature in the field , 44, sarFinally, the remaining 2 cases were difficult to identify as potential pseudoprogression patterns. One patient showed a transient, generalized lymphadenopathy, which constitutes a rather sporadic response pattern under immunotherapy, previously described in some case reports . The sec18F-FDG uptake (SUV) in the spleen and the liver (SLR). Our results showed that SLRmean after the first 2 ICIs’ cycles was significantly higher for patients eventually showing cPMD compared to those with pseudoprogression, suggesting that increased spleen glucose metabolism under ICIs may correlate with negative clinical outcomes. This finding is consistent with recently published literature investigating spleen metabolism by means of 18F-FDG PET, which highlighted the significant association between a high SLR and a poor outcome to immunotherapy [Aside from applying various response criteria, our analysis also involved the semi-quantitative assessment of immune activation signs on PET as a supplementary approach for prediction of the immunotherapy effect. This was attempted through the calculation of the ratio of otherapy , 49. At otherapy , 50, 51,otherapy . On the mean as potential prognosticator in melanoma patients under immunotherapy.Taken together, our findings show that the majority of non-responders can be identified early during immunotherapy, which may carry serious clinical implications. At the same time, approximately 16% of patients with uPMD eventually responds to treatment with remission of signs of pseudoprogression. These results support the use of early PET/CT scanning during immunotherapy, i.e. after application of two cycles of ICIs, but at the same time call for careful handling of the findings of early metabolic progression. In this context, the performance of another follow-up PET/CT at a second time-point for confirmation of early PMD is recommended. Given that the median time required for remission of pseudoprogression was 2.3 months in our cohort, and the fact that a long delay could risk disease decompensation to the point of rendering the patient incapable of receiving salvage chemotherapy, the late follow-up PET/CT examination may be performed approximately 8–10 weeks after initial, early progression. Besides that, we suggest the application of some practical tools for tackling the phenomenon of pseudoprogression, such as the usage of novel metabolic response criteria as well as the identification of sarcoid-like lymphadenopathy as a possible irAE rather than as a manifestation of disease progression. Finally, our results support the supplementary role of SLROur study has some limitations. Firstly, this is a retrospective analysis of prospectively acquired data with a relatively limited number of patients due to the strict inclusion criteria applied. A validation of these findings in larger patient cohorts would be therefore required. Moreover, several patients were not further examined with PET/CT—in terms of the present analysis—after the first 4 cycles of ICIs, since they were either clinically characterized as progressors and subsequently changed/stopped therapy, or they soon thereafter died. Theoretically, a confirmation of the cPMD findings with another PET/CT scan later on (after the 4 cycles), may have revealed remission of signs of pseudoprogression in some of these patients. However, the clinical definition of progressive disease was always predominant, leading to respective, early management changes. Another limitation lies in the fact that most of the patients (84%) described in the present series were treated with ipilimumab monotherapy, which is no longer the standard of care in melanoma; instead, melanoma treatment nowadays involves anti-PD-1 monoclonal antibodies, applied both as single agents and in combination with ipilimumab. Further, the vast majority of the PET/CT-positive findings were not histopathologically confirmed, which is, however, not usually possible in the clinical setting. Finally, this study focused on two sets of criteria, one conventional (EORTC) and one novel (PERCIMT). The other recently proposed, immunotherapy-modified PET response criteria, such as iPERCIST and imPERCIST, were applied only in a subcohort of the studied patient population (pseudoprogression group). However, we are in the ongoing process of evaluating the performance of all available PET criteria—conventional and modified—in an extended patient cohort, including not only patients with PMD but all classes of response to ICIs; these evaluations will be the topic of a future work of our group.18F-FDG PET/CT a cohort of metastatic melanoma patients under ICIs. Approximately five out of six eventual non-responders could be identified with PET/CT already after administration of the first two ICIs cycles, highlighting the potential role of interim PET/CT in early recognition of patients that are unlikely to profit from ICIs. These patients would potentially benefit from an early cessation of the non-effective, potentially toxic treatment and a change in therapeutic management at the appropriate time. On the other hand, 16% of patients with initial signs of metabolic progression was later proven to have pseudoprogression. Therefore, the performance of another follow-up PET/CT approximately 8–10 weeks after initial metabolic progression, in order to tackle the phenomenon of pseudoprogression, is recommended. Moreover, the usage of novel interpretation criteria and the identification of special types of radiological irAEs can further aid in reliable immunotherapy response evaluation. Finally, the investigation of spleen glucose metabolism, as part of the generalized host immune system activation under ICIs, may offer further prognostic information in melanoma patients undergoing immunotherapy.In an attempt to investigate the phenomenon of early metabolic disease progression, we serially monitored with


E. coli with the clear inhibition zone of 200 mm and the detection of bacterial growth showed enhancement with continuous decline throughout the study as compared to free-standing Carv.Recently, the antibacterial properties of Carvacrol (Carv) have been significantly reported. However, due to the unstable properties of Carv under various environment conditions, research approaches tailored towards its widespread and efficient use in various antimicrobial applications are scarce. Here, we discuss progress towards overcoming this challenge by utilising the encapsulation of Carv in gellan gum hydrogels to form thin films (GG-Carv) containing different concentrations of Carv (0.01–0.32 M). FTIR spectrum of GG-Carv revealed that both functional groups from GG and Carv existed. The carbon, hydrogen and nitrogen elemental analysis further supported the encapsulation of Carv with the changes in the element percentage of GG-Carv. Both swelling and degradation percentage increased with time and the decreasing patterns were observed as the concentration of Carv increased. In an antibacterial study, GG-Carv exhibited significant antibacterial activity against  The essential oils from plant materials are potentially useful as a reservoir for antimicrobial compounds. They are well known for having wide applications in folk medicine, fragrance industries, preservation and food flavouring. As recognised in recent reports, essential oils containing compounds such as carvacrol, eugenol and thymol (phenolic compounds) demonstrate high antibacterial potential . The antThymus vulgaris) and oregano (Origanum vulgare). Interestingly, Carv has shown effective antibacterial activity and has been proven to be a potential agent in the treatment of infections, safe for human and animal health [C. albicans [P.fluorescens [L.plantarum, S.cerevisiae, B.cinerea [S.aureus [Clostridium perfringens [Salmonella enterica [L.monocytogenes and E.coli [In this context, we highlight carvacrol (Carv), which is found in the aromatic leaves and flowering plant of both thyme  (>96%). All chemicals were obtained from Sigma-Aldrich . Sodium dihydrogen orthophosphate (NaH2PO4) (≥98%) was purchased from BDH Chemicals Ltd. Poole, UK, sodium hydrogen carbonate (NaHCO3) (≥99.8%) was provided by Fisher Brand , sodium chloride (NaCl) (≥99%) was obtained from AnalaR  and potassium chloride (KCl) (≥99.5%) was purchased from HmbG Chemicals . All chemicals were used directly without any purification. The strain of Escherichia coli , the plasmid of which carries gene resistance against ampicillin, was used in the antibacterial activity study.The chemicals used in this study were glycerine  (>96%), gelzan (gellan gum) (>96%), carvacrol (2-Methyl-5-(1-methylethyl)-phenol) (>98%) and calcium chloride  to a specific concentration and mixed with the dissolved 1 g of gellan gum. This mixture was then mixed with CaClIn this study, GG-Carv was encapsulated with Carv at various concentrations; 0.01, 0.02, 0.04, 0.08, 0.16 and 0.32 M and are hereafter referred as GG-Carv 01, GG-Carv 02, GG-Carv 04, GG-Carv 08, GG-Carv 16 and GG-Carv 32, respectively.−1 on a Perkin-Elmer 1752X Spectrophotometer  with the KBr disc method, in conjunction with the elemental analysis, LECO CHNS-932 Analyser . The surface morphology and the cross-section of sample were observed by using Variable Pressure Scanning Electron Microscopy (VPSEM). This was used for the examination of the samples that are not compatible with high vacuum conditions. The samples were prepared and freeze-dried at −80 °C for 2 days to expel the moisture. Then, the samples were fixed on the aluminium stubs and gold coated before imaging. The model used in this study was LEO 1455 VPSEM .Fourier transform infrared (FTIR) spectra of the samples were recorded in the range of 4000–400 cm3 and 0.35 g of NaH2PO4 were dissolved in 100 mL of deionised water. The solution was stirred until fully dissolved and the resulting solution was then adjusted to pH 5.5 using 10% nitric acid.In this study, Pseudo Extra Cellular Fluid (PECF) buffer solution was used and for the preparation of the buffer, an amount of 0.68 g of NaCl, 0.22 g of KCl, 2.5 g of NaHCOdW) prior to immersion in 20 mL of PECF buffer solution with pH 5.5 at room temperature. The subsequent weight was recorded every 24 h. The films were removed after 72 h, wiped gently with a tissue to expel surface solution and then weighed (wW). The percentage of water uptake was then determined from the equilibrium swelling ratio:wW is the weight of wet samples and dW is the weight of dry samples.Water uptake of GG-Carv with the dimensions of 2 cm × 2 cm was measured by weighing the dried films (iW) and leaving it on a Petri dish at room temperature. The degradation test was performed at room temperature considering to the potential of dermal applications and the common storage temperature. The subsequent weight was recorded every day until a constant weight (fW) pattern was observed. The percentage of degradation was then determined from the equilibrium degradation ratio:fW is the final weight of samples and iW is the initial weight of dry samples.Degradation of GG-Carv was measured by weighing its initial weight of 1.0 g , were also assayed as the negative and positive control, respectively.This study was evaluated by following the disc diffusion test method. The GG-Carv with a diameter of 6 mm (similar to the disc size) was placed onto the surface of the Luria Bertani (LB) agar plate incorporated with ampicillin (Amp) and seeded with 100 µL of E. coli and incubated for 16 h prior to the addition of GG-Carv 08 with dimensions of 10 × 10 mm and 0.08 M Carv. The E. coli culture without any further addition was used as the negative control. The OD of each tube was then measured for every subsequent hour at a wavelength of 600 nm against the standard medium.Here, 10 mL of LB broth incorporated with ampicillin was seeded with 100 µL of E. coli culture. The GG-Carv 08 and 0.08 M Carv was directly placed into the bacterial culture. The OD of each tube was then measured every subsequent hour. The E. coli culture without any further addition was used as the negative control.Here, 10 mL of LB broth with the addition of ampicillin was seeded with 100 µL of Shifting or disappearances of the frequency of functional characteristic peaks indicate the interaction between polymer and drug . The hyd−1 (phenolic–OH group), 2958.46 cm−1 (C–H stretching), 1583 and 1511 cm−1 (C–C ring stretching), 1421 cm−1 (O–H bending), 1359 cm−1 (isopropyl group), 1242 cm−1 (C–O stretching) and 864 cm−1 (aromatic ring). Meanwhile, the peaks of pure GG (−1 (O–H stretching), 2933 cm−1 (C–H stretching), 1625 cm−1 (C=C stretching), 1427 cm−1 (C–H bending), 1033 cm−1 (C–O stretching) and 919 cm−1 (C–H bending).Chemical structures of the samples were characterised by FTIR . In gene pure GG b can be −1 (O–H stretching) and 2887–2933 cm−1 (C–H stretching), which belong to both gellan gum hydrogel and Carv. Furthermore, the peaks at 1637–1642 cm−1 and 1408–1415 cm−1 (C–C ring stretching), 1033–1036 cm−1 (C–O stretching) and 850–918 (aromatic ring), which belong to Carv exist in all GG-Carv samples, reflect the existence of Carv in the gellan gum hydrogel polymer.From the results obtained c–g, all Hypothetically, gellan gum contains the carbon and hydrogen; therefore, to determine the carbon content of gellan gum containing Carv, the resulting carbon percentage will be subtracted from the carbon content of free-standing gellan gum. VPSEM micrographs were used to study the surface and cross sectional area of GG-Carv. The observation was made at 1000 times magnification. This powerful technique is widely used to capture the characteristic “network” structure in hydrogels , encapsuA clear network structure can be observed on the surface morphology of pure GG a. MeanwhUnpacked layers’ structure can be observed in the cross-sectional morphology of pure GG film a. MeanwhHydrogels are three-dimensional, hydrophilic, polymeric networks capable of imbibing large amounts of water or biological fluids . They alThe result showed that the swelling percentage  increaseMost of the degradation study of gellan gum was usually achieved in vivo through the action of enzymes and in vitro  in accorThe percentage of degradation  was founEscherichia coli  strain, the plasmid of which carries gene resistance against ampicillin. A qualitative study was conducted by following the disc diffusion method. Meanwhile, the quantitative study was examined through the detection of bacterial growth by optical density (OD) in two conditions, the antibacterial effect against cell viability and cell growth.We chose to evaluate the antibacterial activities of GG-Carv 08 through qualitative and quantitative studies against the same type of bacteria, E. coli were then compared with the standard antibiotics, with kanamycin as a positive control and ampicillin as a negative control. After 18 h of incubation at 37 °C, results showed that pure GG demonstrated no sign of an inhibition zone  in two conditions, the antibacterial effect against cell viability and cell growth.E. coli followed the normal phase of bacterial growth  was successfully achieved. This was confirmed by the FTIR spectrum of GG-Carv, which showed the combination of both functional groups from the hydrogel and Carv. The elemental analysis further supported the encapsulation with the observed changes to the element percentage. Similarly, the swelling percentage and percentage of degradation increased with the time in normal environmental conditions, but showed a decreasing trend with concentrations of Carv being loaded into the system. GG-Carv showed significant antibacterial activity against


Accumulating evidence emphasizes the relevance of oscillatory synchrony in memory consolidation during sleep. Sleep spindles promote memory retention, especially when occurring in the depolarized upstate of slow oscillation (SO). A less studied topic is the inter-spindle synchrony, i.e. the temporal overlap and phasic coherence between spindles perceived in different electroencephalography channels. In this study, we examined how synchrony between SOs and spindles, as well as between simultaneous spindles, is associated with the retention of novel verbal metaphors. Moreover, we combined the encoding of the metaphors with respiratory phase  with the aim of modulating the strength of memorized items, as previous studies have shown that inhalation entrains neural activity, thereby benefiting memory in a waking condition. In the current study, 27 young adults underwent a two-night mixed-design study with a 12-h delayed memory task during both sleep and waking conditions. As expected, we found better retention over the delay containing sleep, and this outcome was strongly associated with the timing of SO–spindle coupling. However, no associations were observed regarding inter-spindle synchrony or respiratory phase. These findings contribute to a better understanding of the importance of SO–spindle coupling for memory. In contrast, the observed lack of association with inter-spindle synchrony may emphasize the local nature of spindle-related plasticity. The benefits of sleep for memory retention have been widely acknowledged. Sleep protects memory function from wake–time interference and actively consolidates labile traces . Sleep sThe dynamics of temporally overlapping spindles in separate cortical locations is an understudied phenomenon in memory research. The relatively frequent simultaneous or nearly simultaneous occurrence of spindles in more than one electrode location is a consistent finding across studies  and is psaliency of newly potentiated memory traces. A line of research proposes that salient memories are preferably strengthened during sleep  known sleep-dependent consolidation mechanisms  and (2) inter-spindle synchrony, an unstudied potential indicator of widespread memory processing. Our protocol contained delayed retention over both sleep and wake conditions in order to assess if retention (and its association with sleep-related mechanisms) showed differences between these conditions. In addition, motivated by research on respiration and memory, we devised an experimental setting integrating paced breathing into memory tasks in order to examine if encoding-tied respiratory phase affects retention performance. The material in the memory task consisted of novel metaphoric associations . Our maivia different channels; 11 were invited from the previously studied SleepHelsinki! cohort (see details of the cohort1), 15 were students at the University of Helsinki who were contacted via e-mail lists and social media channels within student societies, and three participants were invited through personal contacts. All participants were offered 100 € monetary compensation for their participation. Measurements were performed between June 2020 and January 2021. We screened the participants for learning difficulties and excluded one participant due to a reading disability. We also administered questionnaires for depressive symptoms [Beck Depression Inventory (BDI)]  living in the capital area of Finland. The participants were recruited y (BDI)]  and geney (BDI)]  in ordery (BDI)]  delayed Written informed consent was obtained from all the participants prior to study participation. The study was approved by the Helsinki University Hospital Ethics Committee, and all components of the study were conducted in accordance with the Declaration of Helsinki and its later amendments.We deployed a mixed-design study examining memory outcomes in a within-subjects manner with respect to the delay condition (for sleep and wake conditions) and the respiratory phase . The associations between memory outcomes and sleep oscillation measures were compared between the participants.via Zoom to administer a 10-min nasal breathing exercise, paced by an expanding and shrinking circle on a shared laptop screen . On Day 0 , the parp screen . A polyst-test p = 0.929), with ranges of normative ease of 2.38–5.84 and 2.52–5.90 in the two sets. The presentation order of these sets was counterbalanced. We also balanced subsets of immediate and delayed recall (p = 0.958). For our analyses, we inverted the ease values to represent metaphor difficulty. As an example, the metaphor “(A) malicious remark is a bullet” has lower normative difficulty (2.98) than the metaphor “(A) clever joke is (a) splint” (5.36). The parentheses in the example metaphors denote that there are no articles in Finnish.The memory task was run with Neurobehavioral Systems Presentation software . The task material consisted of two sets of 48 metaphors: one for overnight memory retention and one for over day memory retention. Additionally, each set was divided into subsets of 16 and 32 metaphors as well as immediate and long-delay recall, respectively. The metaphors were chosen based on work on psycholinguistic dimensions , where pOn both occasions of encoding, the participants were shown 48 written metaphors on a laptop screen, with instructions to form a mental image of the metaphors. Each metaphor was shown for 4.25 s within each phase of respiration (see section “Respiratory Phase”), with a 1.5-s interval until the next metaphor was shown. The participants underwent three successive encoding rounds, after which the metaphors were no longer displayed . Thus, the retention and forgetting between immediate and delayed recall could be reliably examined.insect”). Responses were scored so that 1 point was given for a correct word , and 0.5 points were given if the response was a synonym  or a higher/lower abstraction of the correct word . The responses were first scored by two researchers independently and then merged into a combined scale based on mutual agreement on ambiguous responses. The memory retention outcome was calculated so that the percentage of correct responses in delayed recall was subtracted from the percentage of correct responses in immediate recall, resulting in change scores (ΔSleep and ΔWake) corresponding to the delayed condition.The delay from encoding to immediate recall was 10 min (16 metaphors), and delayed recall was 12 h (32 metaphors). In the recall, the participants were shown the beginning of a metaphor on a laptop screen [ touch is (an)”] in random order and were asked to type the missing (last) word  for both inhale-phased and exhale-phased metaphors. Phase-specific retention scores for both delay conditions were calculated separately for inhale- and exhale-phased metaphors by subtracting the delayed recall percentage from the immediate recall percentage.All participants wore Philips Actiwatch 2 actigraphs to screen for highly deviant sleep durations before the laboratory night. The actigraphs were worn for 2 days, between the evening on Day 0 and the evening on Day 2 .All recordings were performed using either SOMNOscreen plus or SOMNOscreen HD . The trained research nurse attached gold cup electrodes at six EEG locations . Electrooculograms (EOGs) and chin electromyograms (EMGs) were measured using disposable adhesive electrodes , with two locations for EOG and three locations for EMG. An online reference (Cz) and a ground electrode in the forehead were used in the current study. The sampling rate was 256 Hz (the hardware filters for SOMNOscreen plus were 0.2–35 Hz). PSG data were scored manually using the DOMINO program  in 30-s epochs into N1, N2, N3 (SWS), REM, and wake according to AASM guidelines . Movement arousals were also marked.The manually scored PSG signals were converted to EDF format in DOMINO software  and then further analyzed using the functions of EEGlab 14.1.2b  running The preprocessed EEG data were further band-pass filtered (order 2816) in the 12- to 16-Hz frequency band. From the filtered signal, spindles were extracted using a method based on an automated detection algorithm described by 2). The signal was first low-pass filtered at 3.5 Hz. All negative and positive amplitude peaks were identified between consecutive positive-to-negative zero-crossings, comprising a full phase cycle. Zero-crossing intervals within the duration of 0.8–5 s were included, corresponding to the 0.2–1.25 frequency range. Finally, mean values for positive and negative peak potentials were calculated, and these events were denoted as SOs where the negative peak was lower than the mean negative peak and where the positive-to-negative peak amplitude difference exceeded the mean amplitude difference. We ran the detection procedure separately for NREM, N2, and N3 sleep.Slow oscillations were detected with an adapted algorithm developed by We operationalized phase-amplitude coupling (PAC) as a modulation index (MI), estimated with an adaptation of the Kullback–Leibler distance proposed by Separately for NREM, N2, and N3 sleep, we calculated the preferred phase (PP) of spindles within SOs . We examined this coupling within each EEG channel. First, we identified spindles where the amplitude peaked within an SO cycle . Next, we band-pass filtered the EEG signal to 0.2–1.25 Hz, Hilbert-transformed the SO signal, and extracted the instantaneous phase at the SO–spindle peaks. The examined variables were SO–spindle% , the circular mean angle in degrees, and the percentage of spindle peaks  occurring at the upstate point (± 45° from 0) for each participant.the seed spindle. (3) Any co-occurring spindle was designated as a trailing spindle. Thus, a seed spindle could occur alone or be overlapped by one, two, or three (different channel) trailing spindles. The direction of the overlap  was recorded. For each electrode pair (seed–trailing), we calculated the propagation latency, overlap duration, and propagation density (PD), which represent the probability of a certain pair  occurring among all seed spindle events in the channel   Proceeding chronologically, when a spindle occurred after a spindle-free period in any channel, we examined if another spindle overlapped  at any other channel. (2) In the case of an overlapping spindle, we examined which spindle peaked first and nominated it as .g., F3) . To incl.g., F3) .signum function, resulting in pair-specific raw PLIs ranging from 0 (random phase difference) to 1 (fixed phase difference). Again, we included spindle pairs that did not overlap instantly (<8 ms). The obtained raw PLIs were further contrasted with randomized spindle-PLIs to highlight the synchrony between overlapping spindles, in contrast to non-overlapping spindles. To this end, the contrast spindle-PLIs were created by calculating the PLI values between each seed spindle and a randomly chosen spindle event from the same participant; this procedure was repeated 100 times. Channel-pair-wise corrected PLIs (cPLIs) were obtained by subtracting the contrast PLIs from the raw PLIs. The cPLIs were categorized according to the direction  for each participant.The phase lag index (PLI)  was compFor memory outcome analyses, we averaged both PD and cPLI values into frontal to central (F–C), central to frontal (C–F), left to right (L–R), and right to left (R–L) means. We only averaged non-diagonal electrode pairs .In the analyses evaluating metaphor difficulty level, one metaphor was considered an observation unit. The association between recall probability in immediate recall and metaphor difficulty was tested using one-way analysis of variance (ANOVA). For delayed recall, we constructed a mixed ANOVA model with recall probability (for both sleep and wake conditions) as dependent variables and constructed metaphor difficulty as a continuous independent variable in order to examine the main effects of metaphor difficulty as well as its interactions with delay conditions.Mean because this variable represents values (degrees) distributed on a circular (not linear) plane.One-way ANOVA was used to test whether the place of measurement was related to sample characteristics  or sex-induced differences in dependent and independent variables. We used repeated-measures ANOVA to test if (1) immediate recall scores differed between evening and morning recalls, (2) if the delay condition affected the delayed retention outcome (ΔSleep and ΔWake), and (3) if the respiratory phase affected pooled immediate recall. The effect of respiratory phase across the delay conditions was tested with a mixed ANOVA model, assigning two levels for phase  and two levels for the delay condition (sleep and wake) as within-subject variables. The associations between memory retention (ΔSleep and ΔWake), SO–spindle coupling variables , and inter-spindle variables (PD and cPLI) were tested using linear regression analyses. A quadratic regression was used to examine the association between memory retention and PPMean values at the group level. We compared the frontal and central NREM PPMean distributions using the Watson–Williams test. The SO–spindle coupling variables in N3 and N2 sleep were compared using a pairwise t-test. The association between PD and cPLI grand mean was tested using Pearson’s correlation. We tested for statistical differences between the variable means using Friedman’s test, and this test was conducted separately for PD and cPLI.Rayleigh’s test of non-uniformity was used to test the circular distribution of PPSex was partialed out from the memory outcome variables in all analyses. In order to evaluate how possible confounders influenced the statistical significance of the results, we also constructed a model controlling for sleep duration, age, BDI score, and GAD-7 score .p < 0.05. Statistical analyses were performed using IBM SPSS Statistics for Windows, version 27.0  or the CircStat Toolbox for Matlab R2018a  as well as higher N3 percentage (p = 0.012) for those who slept at home.F = 6.508, p = 0.017). When evaluating the distributions, we found that one participant showed extremely high values for three SO–spindle coupling variables (>3 SD). This participant was excluded from further analyses.Exploring the dependent and independent variables for any differences related to sex and measurement place (home/laboratory) revealed better retention over sleep in females  but not in immediate recall . The delay condition (sleep/wake) did not interact with difficulty level in terms of recall probability ; that is, the condition did not cause differences in associations between difficulty level and recall probability. Recall probability as a function of metaphor difficulty level is plotted in The mean (standard deviation) recall probabilities for overnight, daytime, and immediate recall conditions were 78.1% (15.1%), 63.1% (18.6%), and 82.5% (11.5%), respectively. The metaphor difficulty level was associated with recall probability in delayed recall . We used a mixed ANOVA with ΔSleep and ΔWake as within-subject variables to test if metaphor retention differed over sleep conditions vs. waking conditions. We found that retention was statistically significantly better over sleep conditions  or delayed recall , nor did it interact with the delayed condition .Within-subject ANOVA of immediate recall performance showed no statistically significant differences between the evening and morning  and central (p < 0.001) SO–spindles for NREM, N2, and N3 sleep. The grand mean degrees (and standard deviations) in NREM sleep for frontal and central SO–spindle PPs, respectively, were 12.7° (20.1°) and 20.9° (17.2°) . Comparison of the coupling measures between N2 and N3 sleep showed that PAC, SO–spindle%, PPMean, and Upstate% were statistically significantly higher in N3 sleep both frontally and centrally  (Assessing the uniformity of PP (17.2°) . Figure ≤ 0.010) .t = −0.111, p = 0.913 and t = −0.129, p = 0.898, respectively) or with ΔWake . No statistically significant associations were found when studying MI in N2 and N3 sleep separately .First, we examined if MI between the oscillation ranges of 0.2–1.25 Hz (phase frequencies) and 12–16 Hz (amplitude frequencies) was associated with the retention of metaphors over sleep and wake conditions. In both the frontal and central derivations, MI over NREM sleep (N2 and N3 combined) was not significantly associated with ΔSleep (p ≥ 0.185). However, we found statistically significant associations with respect to N3 sleep, where the frontal SO–spindle% was statistically significant for ΔSleep . The central SO–spindle% in N3 was statistically significant in the control model . ΔWake was not associated with the N3 SO–spindle ratio .We proceeded to test how the interplay between SO and spindle events is related to delayed memory outcomes. See t = 2.508, p = 0.019) but not central  PPmean angles  and central  derivations, indicating that less forgetting occurred along with an increased probability of spindles peaking at the SO upstate. Running the analyses with the control model did not change the significance status of the results  during N3 sleep, whereas frontal Upstate% showed statistically significant associations during both N2  and N3 sleep   with derivation-specific spindle density and SO number as covariates. All other associations remained statistically significant , but PPMean in N3 sleep was degraded to a trend .Examining how PP over NREM sleep was associated with overnight forgetting of novel metaphors revealed a statistically significant association with frontal (n angles . In addi= 0.016) . FinallyMean, or Upstate% in any sleep stage . To assess the sleep specificity of the observed SO–spindle-coupling in memory retention, we ran a mixed ANOVA with ΔSleep and ΔWake as within-subject variables. The delay condition did not interact with either PPMean or Upstate% .In contrast, ΔWake did not associate significantly with SO–spindle%, PPp-values < 0.001, Bonferroni-corrected), indicating that simultaneous spindles show higher PLI than randomly paired spindles.Investigating how the synchrony of simultaneous sleep spindles related to retention performance included (1) the probability of a seed spindle being overlapped by spindle(s) in other channels (PD) and (2) the degree of oscillation synchrony  between the co-occurring spindles. The mean overlap time across all seed–trailing spindle pairs was 1.003 s. First, we tested whether the channel-pair-wise grand mean raw PLIs differed from the surrogate values and found that they differed significantly  and cPLI . PD ranged between 0.237 (F3–C4) and 0.372 (C4–F4), and cPLI ranged between 0.122 (F3–C4) and 0.198 (F3–C3). The inter-spindle measures are heat-mapped in r = 0.773, p = 0.003), indicating that the probability of propagation between certain electrodes is associated with phase synchrony between electrodes. In For both PD and cPLI, we next examined whether the grand means across the channel pairs differed. Friedman’s test  showed statistically significant differences in PD . The level of corrected PLIs (cPLIs) between co-occurring spindles did not show statistically significant associations with retention over sleep  or waking conditions .In relation to memory retention, we examined PD and cPLI within four mean variables: frontal to central (F-to-C), C-to-F, left-to-right, and right-to-left. With regard to PD, no statistically significant associations were found with ΔSleep or ΔWake  and the PP variables did not indicate sleep specificity on the memory outcome. One possibility for these findings is that coupling accuracy expresses trait-like learning ability. Previous reports suggest that spindle properties are related to immediate learning cognitive abilities , suggestvia corticothalamic modulation, can trigger the matrix pathway within a few oscillatory cycles , was not associated with any of the memory outcome measures. Provided that these overlapping spindles represent “matrix” spindles, our findings do not support their hypothesized role in consolidating widespread memory representations . Howevery cycles . Hence, Acknowledging our inability to fractionate the thalamic origins of EEG spindle events, we focused on the degree of cortical synchronization. To this end, we examined the PLI  between n = 11), warranting replication with a larger sample, it is possible that using paced respiration only during encoding (as in our study) does not evoke statistically significant effects. It is of note that prior evidence on the respiratory benefits on memory  or types of memorized material (recognition). Our results substantiate the accumulating experimental data that highlights the importance of SO–spindle coupling in memory retention.The raw data supporting the conclusions of this article will be made available by the authors on request, without undue reservation.The studies involving human participants were reviewed and approved by Helsinki and Uusimaa hospital district ethics committee. The patients/participants provided their written informed consent to participate in this study.RH: conceptualization, methodology, writing—original draft preparation, formal analysis, investigation, and visualization. LK: conceptualization, review, and editing. MA: data collection, review, and editing. A-KP: conceptualization, writing—review and editing, supervision, and project administration. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.