Expansion of the global protected‐area network has been proposed as a strategy to address threats from accelerating climate change and species extinction. A key step in increasing the effectiveness of such expansion is understanding how novel threats to biodiversity from climate change alter concepts such as rewilding, which have underpinned many proposals for large interconnected reserves. We reviewed potential challenges that climate change poses to rewilding and found that the conservation value of large protected areas persists under climate change. Nevertheless, more attention should be given to protection of microrefugia, macrorefugia, complete environmental gradients, and areas that connect current and future suitable climates and to maintaining ecosystem processes and stabilizing feedbacks via conservation strategies that are resilient to uncertainty regarding climate trends. Because a major element of the threat from climate change stems from its novel geographic patterns, we examined, as an example, the implications for climate‐adaptation planning of latitudinal, longitudinal , and elevational gradients in climate‐change exposure across the Yellowstone‐to‐Yukon region, the locus of an iconic conservation proposal initially designed to conserve wide‐ranging carnivore species. In addition to a continued emphasis on conserving intact landscapes, restoration of degraded low‐elevation areas within the region is needed to capture sites important for landscape‐level climate resilience. Extreme climate exposure projected for boreal North America suggests the need for ambitious goals for expansion of the protected‐area network there to include refugia created by topography and ecological features, such as peatlands, whose conservation can also reduce emissions from carbon stored in soil. Qualitative understanding of underlying reserve design rules and the geography of climate‐change exposure can strengthen the outcomes of inclusive regional planning processes that identify specific sites for protection. Article Impact Statement: Rewilding concepts updated to consider climate change increase mitigation and biodiversity conservation gain from protected area expansion. The twin crises created by the accelerating pace of climate change and loss of biodiversity IPBES  have proSuch proposals are not entirely new. The concept of rewilding, a key element underpinning the call for expanded protected area networks, was defined by Soulé and Noss  as restoAlthough elements of the concept can be found in earlier writings by Victor Shelford and others Croker , rewildiWhile rewilding initially focused on questions concerning landscape structure and reserve design, subsequent development of the concept, especially in Europe, shifted focus toward techniques for restoration of ecosystem processes in formerly human‐modified landscapes  . This qualitative understanding of threat patterns can inform a final stage in which systematic conservation planning methods are used to identify specific sites for protection , a species highly vulnerable to climate change via loss of snow‐covered denning habitat   may be important to persistence of species with modest area requirements under climate change, especially in topographically complex landscapes , wolf, and wolverine . A strong latitudinal gradient in dissimilarity is evident, with more rapid warming in boreal regions than at mid‐latitudes   Fig.  (Hengl eus) Fig. a.Anthropogenic pressure, as represented here by the human modification index  divert resources that would be better spent to protect undeveloped areas irrespective of their climate adaptation value. Although the relative pace of climate versus land‐use change deserves consideration by planners, climate change is projected to alter conservation values even within otherwise undeveloped landscapes and will typically interact with land‐use change to affect biodiversity , are projected to experience extremely high climate exposure  under future warming  for forward climatic connectivity, whereas blue paths indicate areas important for backward climatic connectivity.Click here for additional data file.


All rats were pretreated with ALN (1 μg/kg−1/day−1) for 14 days prior to SFx induction, followed by ALN cessation or continuation for the duration of the experiment; this was combined with daily PTH (8 μg/100 g−1/day−1) on SFx induction for 14 days, followed by cessation or continuation of ALN after SFx induction or an equivalent vehicle as a control. Ulnas were examined 2 weeks or 6 weeks following SFx. Two toluidine blue‐ and two tartrate‐resistant acid phosphatase‐stained sections were examined for histomorphometric analysis using Osteomeasure software. There was a significant interaction between the effects of time and treatment type on the woven bone width and apposition rate, as well as an improvement in the woven bone architecture. However, woven bone variables remained unaffected by the cessation or continuation of ALN. Cessation of ALN increased osteoclast number when compared with the ALN‐PTH continuation group (p = 0.006), and vehicle (p = 0.024) after 2 weeks. There was a significant interaction between the effects of time and treatment type on the number of osteoclasts per unit BMU area and length. The number of osteoclasts per unit BMU area and length was significantly greater in ALN cessation groups. It was concluded that intermittent short‐duration iPTH treatment effectively increased remodeling of SFx with a concurrent BP treatment, provided that BP was ceased at the time of SFx. Our results could help develop shorter iPTH treatment protocols for the clinical management of SFxs and guide clinical decision‐making to cease BP treatment in cases of SFx. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.Parathyroid hormone (PTH) and bisphosphonates (BPs), including alendronate (ALN), have opposing effects on bone dynamics. The extent to which PTH remains effective in the treatment of stress fracture (SFx) in the presence of an ongoing BP treatment has not been tested. SFx was induced in 150 female Wistar rats, divided into five equal groups ( The main difference between SFxs and acute fractures is related to the nature of loading of the bones. An acute fracture typically occurs because of a single maximal loading, whereas SFxs occur because of repetitive submaximal loading. Bisphosphonates (BPs) are stable analogues of pyrophosphatase. They are deposited on bone surfaces within minutes or hours of uptake. The mode of action on the osteoclast is radically different between non‐nitrogen‐containing BPs (first generation) and nitrogen‐containing BPs (second and third generation). Apoptosis is induced by non‐nitrogen‐containing BPs through the formation of a toxic analogue of adenosine triphosphate, whereas nitrogen‐containing BPs (second and third generation) target the enzyme farnesyl diphosphate synthase needed for posttranslational modification of small guanosine triphosphate‐ (GTP‐) binding proteins required for osteoclastic function. All BPs have an affinity for bone tissue, more specifically to osteoclasts, because the acidic pH of resorption lacunae causes an intracellular uptake of BPs, leading to the internalization of substantial amounts of BP. Regardless of the inhibitory pathway, all BPs depress osteoclast activation and resorption.Stress fractures (SFxs) account for about 1% to 7% of all athletic injuries.1, 2 They are incorporated into the bone matrix and have a biological half‐life of more than 10 years. As the duration of antiresorptive treatments increased to 10 years or more, debate arose concerning the risks versus benefits of antiresorptive treatment, and the consequences of “frozen bone.” One of the side‐effects that emerged was a SFx in the proximal femoral diaphysis, which progressed to an atypical femoral fracture (AFF). Recently, a case of fracture in the proximal ulna was also reported after prolonged BP treatment.BPs are highly effective in the treatment of osteoporosis. Numerous large clinical trials demonstrate their efficacy in reducing bone turnover, increasing BMD, and reducing vertebral and nonvertebral fracture risk in patients with osteoporosis.5, 6 It is also acknowledged that initiation of the microdamage is facilitated by bone hypermineralization and accumulation of advanced glycation end‐products that reduce the toughness of the bone matrix. This hypothesis is supported by the location of the AFF in areas of stress , and the fact that such fractures develop over a long period and are preceded by episodes of prodromal pain. There are a lot of similarities between SFxs and AFFs in terms of etiology and possible management protocols. Therefore, we hypothesized that anabolic PTH treatment would accelerate SFxs, even in the presence of BP treatment.It is now well‐accepted that the pathogenesis of AFF is its initiation as a SFx caused by an accumulation of unrepaired microdamage following suppression of bone turnover by BPs.16 It  Intermittent administration of PTH results in increased bone formation, and cortical bone volume and width. It also improves bone architecture and mechanical properties in different species. The potential for PTH to treat localized osseous defects and accelerate SFx healing has also been investigated. Conversely, combination therapy of PTH plus BP  impairs the anabolic function of PTH and its ability to enhance BMD. For example, PTH treatment for 2 months after an antiresorptive agent increased bone formation and the mineralization rate, but it was less than that achieved with PTH treatment alone, though this observation has not been universal. So the question remains as to what extent PTH can remain effective in the presence of BP treatment.Many studies confirm the anabolic effect of PTH and its correlation with the timing and duration of treatment.25 Int However, formation of a periosteal callus was not adversely affected. This woven bone reaction acts to return the bone to its original strength 2 weeks after SFx induction. However, there is little analysis available for the combined effects of a BP with PTH. In the ulna SFx model, PTH increased BMC significantly by 7% at 4 weeks and BMD and BMC significantly by 10% and 7% at 8 weeks compared with controls, whereas ALN did not change BMD or BMC. PTH significantly stimulated bone formation by 114% at 2 weeks, increased intracortical resorption area by 23% at 4 weeks, and enhanced the ultimate force of the affected ulnas by 15% at 8 weeks. Similar to Kidd and colleagues, ALN significantly suppressed the bone formation rate by 44% compared with the control at 4 weeks. These data suggest that PTH could accelerate SFx remodeling, but the combined effects of PTH with BPs remain unknown.We previously showed that BP treatment (risedronate) impaired healing of a SFx by reducing the volume of bone resorbed and replaced during remodeling.38 How Patients receiving long‐term BP treatment may be at higher risk for development of SFx, and more serious complications including AFF. Although some studies have investigated the effect of combined PTH‐BP therapy in osteoporosis, this is not the case for SFx and potential AFF. Therefore, the aim of the current experiment was to investigate the efficacy of a daily iPTH treatment for 14 days in acceleration of SFx remodeling and healing indices in the rat ulna in the presence or absence of a concurrent BP treatment (ALN).The ulnar loading model in rats provides an effective approach to examine focal SFx remodeling with a known time course and precise anatomical location.42 PatThe Griffith University Animal Ethics Committee  approved the experimental protocols (GU Ref No: MSC/02/13/AEC). Accounting for variability, anesthesia, and recovery, four to five rats were loaded/day. Loading was performed in a loading device using a Linear Voltage Displacement Transducer (LVDT) to monitor displacement in the limb. Because of the natural ulnar curvature, axial compression was converted into bending forces with the lateral cortex in tension and the medial in compression. Loading involved cyclic compressive loading at 18 to 20 N load and 2‐Hz cycle frequency. Loading was stopped at a predetermined point when increased displacement reached 10%. As noted above, this level of stiffness loss consistently produced SFxs. A single injection of opioid analgesia (buprenorphine 0.05 mg/kg s.c.) was used following loading sessions.We induced an ulnar SFx in 150 female Wistar rats, 12 weeks of age, weight (300 ± 15 g). Rats were anesthetized with halothane and oxygen for loading. SFx was achieved in a single loading session of the forearm in axial compression at 2 Hz until an increase in displacement of 10% was reached, on average after approximately 8000 cycles or about 60 minutes . This produced a remarkably standardized SFx.31 Accn = 15 each). PTH‐treated groups received s.c. injections of human PTH‐(1–34) peptide  dissolved in 0.9% saline with 1% rat heat‐inactivated serum in a final volume of 200 μL with a dose of 8 μg/100 g/day. PTH treatment was started 24 hours after SFx loading and continued daily for 14 days. ALN‐treated groups received s.c. injections of ALN in saline carrier, once daily at 1.0 μg/kg/day. The ALN treatment started 14 days before the SFx loading. The ALN dose was adjusted for the metabolic rate of the rat and equivalent to the human clinical dose. VEH groups received an equivalent dose of saline for each ALN injection and an equivalent dose of rat serum for each PTH injection in the corresponding treatment groups. Rats were euthanized at 2‐ and 6‐weeks post‐SFx , and vehicle‐ (VEH) treated groups Cortical bone area 2)Woven bone area (mmWoven bone perimeter (mm)Woven bone width (Wo.B.Wi [mm])Length of SFx (μm)Length of remodeling unit along the microcrack (μm)2)Porosity BMU area (μmPorosity BMU area perimeter (μm)2)Erosion  area  perimeter (μm)2)Healing area Number of osteoclastsOsteoclasts surface perimeter (μm)Two toluidine blue and two tartrate‐resistant acid phosphatase‐stained sections were examined for histomorphometric analysis using Osteomeasure software . As previously described, histomorphometry was performed at a standard level along the SFx Fig. A, whereles Fig. :Cortical2 of BMU area (number of osteoclasts /μm2)Number of osteoclasts per μmNumber of osteoclasts per μm of BMU length (number of osteoclasts/μm)2]/porosity BMU area × 100 [μm2])Healing percentage From the standard variables measured above, the following derived variables were obtained:Percentage fracture length occupied by bone formation 2 of the cortical bone area Healing area per mm2 of the cortical bone area (porosity BMU area [μm2] /Ct.Ar [mm2])Porosity BMU area per mmPercentage fracture length occupied by erosion 2 of the cortical bone area Erosion area per mmIn addition, the following derived variables were calculated to correct for variations in the total length of the microcrack.p ≤ 0.05.The data collected from this experiment were analyzed using a two‐way ANOVA with time and treatment group as the independent variables. In the presence of a significant statistical interaction, the main effects in the original two‐way ANOVA were ignored, post hoc pairwise comparisons were performed between individual groups and differences determined using Fisher's least significant difference. In the presence of a nonsignificant statistical interaction, the ANOVA main effect for each independent variable (time or treatment group) was reported independently. Significance was accepted at 2), and the length of SFx (μm).There were no significant differences among groups in terms of Ct.Ar and cortical bone perimeter, woven bone area (mm1 versus ALN2 versus ALN‐PTH1 versus ALN‐PTH2 versus VEH) on Wo.B.Wi . Continuation or cessation of ALN had no effect on Wo.B.Wi 2 weeks post‐SFx induction. Wo.B.Wi was significantly higher in the combined ALN‐PTH1 treatment group when compared with the ALN1 (p = 0.006) group after 2 weeks. It was also significantly higher in the combined ALN‐PTH2 treatment group when compared with the ALN2 (p < 0.001) group at the same time frame. After 6 weeks, Wo.B.Wi was significantly greater in the continuous ALN‐PTH1 treatment group when compared with ALN cessation in ALN‐PTH2 treatment (p = 0.05) group and the VEH group  on the woven bone apposition rate . The woven bone apposition rate was significantly greater in the combined treatment groups (ALN‐PTH1 and ALN‐PTH2) when compared with the ALN treatment groups (ALN1 and ALN2) after 2 weeks of SFx induction . Furthermore, the woven bone apposition rate was significantly higher in the ALN‐PTH1 and ALN‐PTH2 groups when compared with the VEH group after 2 weeks of SFx induction . Finally, the woven bone apposition rate decreased significantly in all groups after 6 weeks when compared with the second week  and VEH groups  on the number of osteoclasts per unit porosity BMU area . The number of osteoclasts per unit porosity BMU area was significantly greater after 2 weeks following cessation of ALN (ALN2), when compared with continuous ALN1 treatment (p < .001). Furthermore, the number of osteoclasts per unit porosity BMU area was significantly greater after 2 weeks of ALN cessation in the ALN‐PTH2 group when compared with when ALN treatment was continued in the ALN‐PTH1 group  when compared with the ALN continuation groups  on the number of osteoclasts per unit BMU length . The number of osteoclasts per unit BMU length was significantly greater 2 weeks after ceasing ALN (ALN‐PTH2 group) when compared with ALN continuation in the ALN‐PTH1 group , the ALN2 group (p = 0.001), and the ALN‐PTH2 group (p < 0.001).There was a significant interaction between the main effects of time and the type of treatment , and the VEH  phase of the remodeling cycle, whereas 6 weeks is the most representative time point for the later (formation) phase of remodeling.Our results can be explained by understanding the mechanism of healing of SFx, which occurs through direct remodeling and starts from the periosteal exit point of the SFx and progresses along the SFx line. BMUs are formed in the first 2 weeks, and osteoclasts play a significant role at this initial stage of healing.22 Thi which is approximately 4 weeks in a rat's life span. We reduced the treatment window of PTH to the shortest possible period through administration of 14 daily injections, which is sufficient to induce stable (plateau) serum PTH levels. The significant increase in osteoclast number along the SFx was expected at 2 weeks following PTH treatment. This may be explained by the PTH induction of monocyte chemotactic protein‐1, which is responsible for differentiation and recruitment of osteoclast precursors in early remodeling phases. The mechanism of the anabolic action of PTH is also supported by other factors, including calcium availability and the calcium sensing receptor. The downregulation of sclerostin has also been linked to the anabolic PTH effect, through reduction of Sost expression, which negatively regulates Wnt signaling.We also pretreated rats with ALN for 14 days, after which rats were treated with PTH for 14 days in the ongoing presence of ALN or its cessation. Current PTH treatment protocols are 18 to 24 months,46 whi For example, greater bone turnover was achieved in women with osteoporosis receiving antiresorptive therapy (ALN) for 18 months after cessation of that therapy and switching to PTH for an additional 18 months. It was suggested in a previous study that the additive effect of the combined ALN‐PTH treatment is attributable to increased bone formation with a combined osteoclast inhibitory action, implying that the anabolic effect of PTH might be independent of resorption. Furthermore, in cases of combined treatment, decreasing the frequency of an antiresorptive agent administration results in an increase in the overall BMD. It was also suggested that pretreatment with PTH, followed by an antiresorptive agent like ALN and continuing the combination therapy could improve treatment outcomes. However, although this could be applied to chronic conditions such as osteoporosis, it is not feasible for SFx, where the timing of injury cannot be predicted beforehand to allow for sufficient time for pretreatment with PTH.Our study is the first study to investigate the combined effect of intermittent PTH treatment with a concurrent or ceased ALN treatment on the overall remodeling of SFx. There have been inconsistent outcomes with regards to the combined antiresorptive and PTH therapies in the treatment of postmenopausal women with osteoporosis.59 For The optimal length of a drug holiday has not been established, but existing data suggest 3 to 5 years with ALN, 3 to 6 years with zoledronate, and 1 to 2 years with risedronate. A decision to recommence therapy should then probably be based on regular reassessment of BMD and fracture risk. New alternative drugs with a shorter duration of action like denosumab could provide a more reversible option as an antiresorptive agent and could potentially be beneficial for patients with SFxs to prevent the risk of fractures, or at least allow for a quicker healing of an AFF caused by earlier activation of remodeling. Romosozumab , developed as a humanized monoclonal antibody against sclerostin, could also support the formation phase of remodeling.Contrary to our hypothesis, intermittent PTH treatment was more effective in SFx repair when ALN treatment was stopped. This is based on the greater recruitment and availability of osteoclasts that initiate the resorption phase of bone remodeling. Although the percentage healing of the SFx was greater following cessation of ALN, it was not significant at 6 weeks in this experiment. The current clinical treatment protocols for osteoporosis are based on long‐term drug administration with chosen periods of cessation called “drug holidays.” The evidence for the value of the drug holiday periods remains weak, and a careful evaluation for the risks versus benefits of cessation of BPs should be investigated thoroughly before making a decision.61 The Limited data are available from BP discontinuation clinical trials, but suggest a residual and continuous biological effect of BPs, especially ALN and zoledronic acid for a lengthy period after discontinuation. It was hypothesized that BP treatment causes tissue brittleness that initiates cracks, increases homogeneity of osteonal and interstitial structures, impairs targeted repair by BMUs, and allows easier accumulation of microdamage at areas of maximum force and mechanical loading.BPs are known to have a long residence time in bone, and to also be recycled and return to other resorptive surfaces even after the treatment has ceased.65 Lim There is debate as to whether the improved healing of AFF was because of teriparatide treatment, the discontinuation of BPs, or both factors combined. Furthermore, a reduction in the incidence of AFF by 70% per year was reported, when BPs were ceased. This was attributed to the osteoclastic bone resorption and repair of microcracks that follows the discontinuation of BP treatment. iPTH has shown promising results in the treatment of BP‐related AFFs. In one of the case reports, despite successful healing of AFF using teriparatide, the authors questioned that it had a major role in the healing process. It was hypothesized that surgical fixation, vitamin D therapy, calcium, and ALN discontinuation could have played a secondary role in the healing process. This aligns with the results from the current study where greater remodeling activation was observed following cessation of ALN.In clinical studies, healing of nondisplaced AFF improved when BPs were ceased.68 The This illustrates the difficulty of managing cases of SFx in association with antiresorptive therapy. That is, cessation of antiresorptive agents is favored for healing of SFx, but compromises the structural integrity of bone in osteoporotic patients, whereas continuation of antiresorptive treatment may delay healing and lead to atypical fractures. In the absence of randomized controlled trials on this question, a taskforce of the American Society for Bone and Mineral Research recommends discontinuation of BPs, adequate calcium, and vitamin D, and consideration of teriparatide for those who appear not to heal on conservative therapy. The above‐mentioned results highlight the importance of the current findings related to SFx healing and note the variability of treatment outcomes as a limitation that could be related to the different animal model or unique clinical scenario of each individual case, as well as the different treatment protocols adopted in each study.In a recent case report, discontinuation of BPs and treatment with teriparatide resulted in a nearly complete radiographically verified healing of AFF, but unfortunately 12 months after the cessation of BPs, bilateral recurrence of AFF occurred, despite sequential use of teriparatide and denosumab.75 Thi and dispels the questions concerning efficacy of PTH in the formation of new bone around sites that have been previously suppressed by BP treatment. For example, PTH therapy can enable new bone formation and replace the old bone matrix on a previously resorption‐suppressed site caused by BP treatment. Furthermore, PTH when compared with other anabolic agents like strontium ranelate, has a long‐term stimulating effect on bone formation and resorption markers in postmenopausal women with osteoporosis previously treated with BPs.The higher values for woven bone parameters observed in this study is consistent with others,76 and The efficacy of PTH in the formation of new bone around sites that have been previously suppressed by BP treatment had been questioned. Later, it was demonstrated that PTH could enable new bone formation and replace the old bone matrix on a previously resorption‐suppressed site following BP treatment. The increase in healing parameters in the ALN‐PTH group following cessation of ALN was greater, but not statistically significant, at 6 weeks. Ma and colleagues did show that cortical bone formation was lower in ALN‐pretreated groups when compared with PTH‐only groups. This was explained by the greater intensity of resorption during remodeling activation that leads in sequence to greater formation. The earlier increase in osteoclastic activity was certainly observed in this study.The present study showed that the anabolic PTH action was not affected or inhibited by prior ALN treatment. However, continuation of ALN treatment with a concurrent PTH treatment depressed its anabolic effect, and the increased Wo.B.Wi was consistent with other studies.76 The This opens the possibilities for investigating the effects of weekly rather than daily PTH injections, which has been investigated previously, but only in the treatment of AFF. It also highlights the different effects on bone surfaces, including the woven bone callus, and the remodeling required to heal the SFx line.Our results show that after 6 weeks, daily PTH injections for 14 days did not elicit the same effect, consistent with activated modeling (formation). This is in alignment with a recent study that showed a higher bone volume with higher daily PTH doses as a result of a higher trabecular number in the healing callus and a denser trabecular bone network, indicating increased bone formation.80 ThiIt was concluded that cessation of ALN after SFx induction in the combined ALN‐PTH treatment was effective in increasing osteoclast parameters. Daily iPTH injections for 14 days improved the woven bone apposition rate and osteoclast parameters. Other woven bone and healing parameters remained unaffected by the cessation or continuation of ALN. It is possible that relatively short periods of iPTH therapy could activate remodeling of SFx following treatment with BPs, as long it is ceased at the time of SFx induction.The authors declare no conflicts of interest.Mahmoud Bakr: Conceptualization; data curation; formal analysis; investigation; methodology; project administration; validation; writing‐original draft; writing‐review and editing. Wendy Kelly: Data curation; investigation; project administration; writing‐review and editing. Athena Brunt: Data curation; investigation; methodology; project administration; writing‐review and editing. Bradley Paterson: Data curation; investigation; methodology; project administration. Helen Massa: Data curation; investigation; methodology; supervision; validation; writing‐review and editing. Nigel Morrison: Conceptualization; formal analysis; funding acquisition; investigation; methodology; supervision; validation; writing‐review and editing. Mark Forwood: Conceptualization; formal analysis; funding acquisition; investigation; methodology; project administration; supervision; validation; writing‐review and editing.


The WHO estimates there are 5.6 million new cases of syphilis annually.At the STI clinic in Amsterdam, we were recently confronted with a treatment failure in a patient with syphilis who was treated with benzylpenicillin (BP) intramuscular injections by his general practitioner (GP). In a second case, we were consulted by a GP who considered a treatment with BP. In both cases, the GP was advised by the pharmacist to administer intramuscular BP as an alternative treatment for syphilis. Also, the national pharmacist information system provided confusing advices on the alternatives for BBP treatment.BP is a short-acting antibiotic, usually administered intravenously for the treatment of neurosyphilis and congenital syphilis in daily dosage regimes for multiple days. BBP is a salt form of BP stabilised with the benzathine group. When administered intramuscularly, BBP is slowly hydrolysed into BP and thus works as a long-acting depot penicillin.BBP successfully cures uncomplicated early syphilis infections  with a single dose of 2.4 million units intramuscular. For the late latent and unknown duration syphilis stages, weekly 2.4 million units intramuscular for 3 weeks is the recommended regime. Under no circumstances can intramuscular BP replace intramuscular BBP following the same dosage regimes for the treatment of uncomplicated syphilis.Duration of treponemicidal level of antimicrobials should be at least 7–10 days to cover a number of division times (30–33 hours).In the absence of BBP, doxycycline 100 mg two times per day for 14 days can be used as an alternative for the treatment of early uncomplicated syphilis and 100 mg two times per day for 28 days for the late latent stage and stage of unknown duration syphilis. Strict serological follow-up is recommended when treating with doxycycline to exclude treatment failure. Although the names are quite similar, physicians and pharmacists need to be aware of the differences in the working mechanism of BP and BBP to avoid using inappropriate treatments of infectious syphilis.


Metal nanoparticles (NP) that exhibit localized surface plasmon resonance play an important role in metal‐enhanced fluorescence (MEF) and surface‐enhanced Raman scattering (SERS). Among the optical biosensors, MEF and SERS stand out to be the most sensitive techniques to detect a wide range of analytes from ions, biomolecules to macromolecules and microorganisms. Particularly, anisotropic metal NPs with strongly enhanced electric field at their sharp corners/edges under a wide range of excitation wavelengths are highly suitable for developing the ultrasensitive plasmon‐enhanced biosensors. In this review, we first highlight the reliable methods for the synthesis of anisotropic gold NPs and silver NPs in high yield, as well as their alloys and composites with good control of size and shape. It is followed by the discussion of different sensing mechanisms and recent advances in the MEF and SERS biosensor designs. This includes the review of surface functionalization, bioconjugation and (directed/self) assembly methods as well as the selection/screening of specific biorecognition elements such as aptamers or antibodies for the highly selective bio‐detection. The right combinations of metal nanoparticles, biorecognition element and assay design will lead to the successful development of MEF and SERS biosensors targeting different analytes both in‐vitro and in‐vivo. Finally, the prospects and challenges of metal‐enhanced biosensors for future nanomedicine in achieving ultrasensitive and fast medical diagnostics, high‐throughput drug discovery as well as effective and reliable theranostic treatment are discussed. Plasmonic nanostructures with anisotropic shapes possess dramatic enhanced electric field when they interact with light. This unique feature has led to the design of metal‐enhanced fluorescence (MEF) biosensors and surface‐enhanced Raman scattering (SERS) biosensors with ultrasensitivity, where metal nanostructure plays an important role as plasmonic enhancer to amplify the optical signals for biological detection. For the development of nanoparticle‐based biosensors , surface functionalization of metal nanostructures plays very crucial role to enable target specific detection. Thus, different biofunctionalization and assembly methods in preparing the bio‐metal sensing nanoprobes will be discussed. Subsequently, the designs and applications of metal‐enhanced biosensors will be reviewed, which include the recent progress in both planar and colloidal NPs‐based MEF and SERS biosensors. Particularly, the planar‐based MEF and SERS systems were found to be more sensitive than the colloidal biosensors, as aggregation of metal NPs on planar substrate can led to a stronger enhanced electric field in the gap area to form hot spots for detection. Moreover, colloidal biosensors are more suitable for in‐vivo sensing due to the small size of colloidal NPs in aqueous solution similar to most of the biological molecules and cells under physiological conditions. Thus, colloidal biosensors can also be developed into multifunctional probes for theranostic treatment combining diagnostic and therapeutic functions in one application. The success of metal‐enhanced biosensors for future nanomedicine relies on the accomplishment of various technical challenges ranging from theoretical study of metal enhancement effects to standardization of biosensor fabrication methods, as well as further improvement in assay design to achieve single molecule detection for in vivo applications. In addition, development of biocompatible metal‐enhanced biosensors with minimum signal‐to‐noise ratio is of utmost importance for clinical translation, especially for medical diagnostics, drug discovery, and theranostic treatment.22.1Gold nanoparticles (AuNPs) of different size and shape could be synthesized under specific reaction conditions.2.1.1+ ions  precursor, cetrimonium bromide (CTAB), trace amount of Ag+ ions, and ascorbic acid (AA). While CTAB forms the cylindrical micelle  to create a nanoreactor for the formation of rod‐shape seed, adding trace amount of Ag+ ions to the reaction mixture is important to facilitate the longitudinal growth of AuNRs. In addition, AA allows the slow deposition of Au+ ions onto the Au seed, preventing fast nucleation that is unfavourable for the controlled growth of Au nanorods. The as‐synthesized AuNRs normally exhibit the penta‐twinned structure with {111} faces at their two ends and {100} or {110} along the longitudinal faces.+ ions (using AgNO3 as the precursor) would enable the high yield synthesis (>95% yield) of single‐crystalline AuNRs with the aspect ratios less than 5.4 as a strong reducing agent. The aspect ratio can be controlled between 2 and 5 by adjusting the chemical reagents‐to‐gold precursor ratios  and pH of the reaction mixture. It is worth noting that the synthesis of AuNRs is very sensitive to the impurity present in the precursors. For example, using CTAB obtained from different manufacturer or lot number for the AuNR synthesis might result in variation of particle quality from batch to batch production. It has been shown that the impurity  in CTAB resulted in the failure synthesis.Gold nanorods (AuNRs) are one of the most popular shapes among the anisotropic AuNPs, due to their well‐established synthesis protocols to control the size, aspect ratio, and monodispersity. Apart from the pioneering works on hard‐templating2.1.24, HAuBr4, and AuCl− and I− or Ag+),4 in affecting the shape of Au polyhedral nanostructures via the seed‐mediated growth approach. Results shows that higher concentration of Au ions led to the formation of Au nanostructures from octahedral to rhombic dodecahedra and cube , AgNO3 , HCl , and ascorbic acid  for overnight aging. The sizes of Au bipyramids can be controlled by the amount of added Au seeds.2O2Gold bipyramids nanoparticle (AuBPs) are the exceptional nanostructures with promising catalytic and optical properties because of their six {110} facets, which has a higher surface energy than other facets such as {100} and {111} facets,2.1.44, CTAC, iodine ions and ascorbic acid to synthesize monodispersed triangular Au nanoplates at pH 8  could produce tri‐iodide ions (I3−) in the presence of oxygen molecules, where the I3− ions act as the strong chemical etchant to selectively remove the spherical and irregular shapes at an early growth stage under alkaline conditionAu nanoplates are plate‐like nanostructures with two {111} facets exhibiting unique optical properties which can be tuned from visible to NIR region by careful control of their edge length, thickness and morphology .2O2 at defined ratio  to etch the corners of Au nanoplates. The thickness and diameter of the circular Au nanodisc could be tuned in the range of 10–50 nm and 50–150 nm, respectively. Figure Numerous literatures have reported the fabrication of Au nanodiscs using physical methods such as electron‐beam lithography.2O2.On the other hand, star‐shape Au nanoplates can be synthesized using mild reducing agent e. g., ascorbic acid, in the presence of shape–driving agent such as polyvinyl pyrrolydine (PVP)2.1.5Multi‐branch/spiky gold nanostructures have received tremendous attention due to the strongly enhanced electric field at their tips, enabling red to NIR adsorption with good photothermal effects. Generally, there are two main strategies for the synthesis of gold nanostars, i. e., seed‐mediated growth and seedless approaches, both of which using different chemicals to induce the deposition of Au ions on Au nuclei in different directions.+ or Fe2+) or using surfactants with multiple moieties. This method usually results in the formation of Au nanostars, which contain a spherical core surrounding with multiple tips. The number of tips could be varied from a few to uncountable number of tips .In seed‐mediated growth approach, the anisotropic growth of Au nanostar is induced by the addition of another metal ions  of Au nanostars could be obtained in a reaction with citric acid, hydrogen peroxide and bis(p‐sulfonatophenyl) phenylphosphine dihydrate dipotassium as stabilizer.Another seedless synthesis approach is based on the use of “good buffers” which act as both reducing agent and shape‐driving capping agent for the one‐pot synthesis of Au nanostars. In this approach, the type of buffer molecule, buffer‐to‐Au ions molar ratio and pH of reaction mixtures all play important role in determining the properties of the final product. To date, many studies have reported the successful synthesis of Au nanostars using good buffers, such as 4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid (HEPES), 3‐(N‐morpholino)propanesulfonic acid (MOPS), and 4‐(2‐hydroxyethyl)‐1piperazinepropanesulfonic acid (EPPS)). Among these “good buffers”, only HEPES could lead to the formation of Au nanostars with asymmetric tips in high yield (>95%)2.22.2.13 and ascorbic acid, followed by addition of NaOH solution. The sizes of Ag nanorods were tuned by varying the amount of Ag seeds.+ precursor, reaction time and amount of Ag seeds in the presence of citrate and PVP in water.The synthesis of Ag nanorods was first reported by Murphy's group, where 4 nm citrate‐capped Ag seeds2.2.23COOAg as the silver precursor, ascorbic acid as the reducing agent, and also Fe3+ ions as the shape driving agent to obtain Ag nanocubes with sharp edges  and corners. The growth of cubic structure within the AgCl octahedral formed as a result of mixing CTAC and CF3COOAgThe synthesis of silver‐based polyhedral NPs, particularly Ag nanocubes, relies on the selection of shape‐driving agents to be adsorbed on specific facets of Ag seeds  and guide the direction of growth into Ag polyhedral NPs with well‐defined shape. PVP and halide ions are the most important shape‐driving agents for the synthesis of Ag polyhedral nanostructure.2.2.3+ ions in the presence of phenylphosphine dihydrate dipotassium salt (BSPP). However, photo‐mediated approaches require a relatively long irradiation time . More recently, our group have developed a new seed‐mediated approach in aqueous media via the selective growth cum oxidative etching.2+ and ascorbic acid react with the oxygen molecules existed on {111} and {110} facets of Ag seeds, resulting in the in‐situ formation of H2O2 which etches these facets. On the other hand, CTAB capping agent facilitates the growth of {100} facets due to its higher affinity (Br−) binding to the {100} facets, which protect them from oxidative etching. The whole reaction takes only 2 hours, which is much shorter than previously reported methods in forming AgBPs . Typically, the reported procedures for the synthesis of AgBPs requires rather long reaction time  or involves the use of organic solvent, high temperature, and ultra‐pure chemicals for the high yield synthesis. For example, Wiley et al.s Figure d.2.2.4N,N‐dimethyl formamide (DMF) as both organic solvent and reducing agent.+ ions. However, large amount of multiple twinned particles (MPTs) was obtained as a by‐product.via O2‐mediated Ostwald ripening  in g Figure e, top.Photo‐mediated aqueous synthesis is also used to obtain silver nanoplates from Ag seeds in the presence of PVP and citrate.3+ or Ag3) exist in the nucleation stage of a solution‐phase synthesis using AgNO3 as the precursor.+ ions in the solution, slowing down the reduction rate substantially to form the thin plates.Besides, Xia's group also reported the synthesis of Ag nanoplates by manipulating the PVP/Ag+ ratio and molecular weight of PVP in the absence of citrate. This study indicated the dual‐functional roles of PVP as both reductant  synthesis of core and ii) growth of shell. The important parameters of the synthesis include mild reducing condition  for growing the shell and adjusting the ratio of the metal core (as seeds) to metal ions (as precursor for shell growth).2.3.22.3.2.1+ ions and Au3+ ions usually occur simultaneously. By varying the ratio between the two metal precursors, the compositions and thus the optical properties of the final AuAg alloy nanoparticles could be tuned between the absorption wavelength of the pure AgNPs and AuNPs in the visible region.Co‐reduction method have been mostly used to develop spherical AuAg alloy nanoparticles,2.3.2.23++3e−→Au, E=+1.498 V) and silver .4). Through the careful control of the reaction time, this method can lead to formation of alloy AgAu nanostructures, which are mostly hollow.Galvanic replacement method relies on the difference between the reduction potential of gold (Au2.3.2.34OH)  to detect specific target biomolecule. The biomolecular interaction can also be used to assemble the optical probes  and metal NPs to form the sensing probes for MEF and SERS. In the following session, we will discuss the different strategies to functionalize the surface of metal nanoparticles for metal‐enhanced biosensors.Capping agents are usually used in the aqueous synthesis of anisotropic metal nanoparticles to stabilize and direct the growth of anisotropic metal nanoparticles. In some cases, the capping molecules  are loosely bound to the surface of metal NPs which could interact with other charged molecules 3.1Biofunctionalization, which enable biological recognition on metal nanoparticles, is one of the most important aspects in designing metal‐enhanced biosensors. Affinity binding in the biological systems often acts as the “lock and key”, where specific pair of biomolecules such as antibody‐antigen,These biorecognition elements are not only important for target‐specific binding, but also useful in particle stabilization and to direct the assembly of nanoparticles for biosensor development. For example, our groups have developed several colorimetric and DLS ‐based nanoparticle biosensors using different biorecognition‐induced aggregation mechanisms to detect various analytes from small drug molecules, DNA/RNA, protein biomarkers to cancer cells.3.2Among the aforementioned biorecognitions, DNA hybridizations is often employed to direct the metal nanoparticle assembly for the plasmonic biosensors design based on the aggregation‐induced solution color  or size changes .3.3Besides biorecognition, non‐specific interactions such as electrostatic attractions can be exploited for nanoparticle assembly. Particularly, layer‐by‐layer assembly (LBL) technique has been widely used to build up layers of polyelectrolytes on either planar surfaces or surface of metal nanoparticles (NPs). This method uses electrostatic interactions between differently charged polyelectrolytes to assemble different number of bilayers on the substrate or NP surfaces. The self‐assembly process of each pair of polyelectrolyte bilayers can be repeated until reaching the desired thickness on surfaces. LBL assembly can be employed to control the distance between metal nanoparticle and fluorophore in the MEF biosensor.44.10) to the excited singlet state (S1). As illustrated in the Jablonski diagram in Figure When a classical fluorophore is excited under specific excitation wavelength, its electrons go from the ground state  between the fluorophore and metal NPs to achieve the maximum fluorescence enhancement factor.As MEF is a distance‐dependent phenomenon, precise control of the separation distance between metal nanoparticle and fluorophore (or gap) is an important issue in designing biosensor. This gap is usually made by a dielectric material. The dielectric layer could be silica, polymer or even biomolecules that have been discussed extensively in the literature.4.2There are two types of MEF biosensors based on the use of metal nanostructures in either planar or colloidal systems. Table 4.2.1One of the most popular methods for developing MEF biosensors is the deposition of pre‐synthesized metal nanoparticles on the surface of a planar substrate through either electrostatic interaction or chemical bond. The planar substrates include hard‐substrate  and flexible materials . As mentioned previously, the distance between the metal NPs and fluorophore plays an important role to the fluorescence enhancement. Typically, this distance can be controlled by the recognition elements themselves  or through the addition of a secondary dielectric layer on the metal NPs surfaces before conjugation with the biorecognition element. Depending on the biosensor design, it is also possible that the analyte molecule itself act as the fluorophore in the MEF system.Metal‐enhanced fluorescence has been extensively studied using glass slides as the substrate. The glass slide should be cleaned by either aqua regia solution or light treatment (UV light and UV ozone) or plasma cleaner. This cleaning procedure is essential to remove any organic substance from the surface and make it more hydrophilic with surface charges for subsequent modification. It is easy to deposit the metal NPs onto the glass surface through electrostatic interactions or silane chemistry to form the chemical bond. After which, the metal NP‐deposited glass slide can be functionalized with the biorecognition elements  to develop the sandwich structure for biological detection. Camacho et al.4.2.2section 4.2.3).Developing colloidal MEF biosensors is of great importance since most of the biological phenomena occur in the solution at the nanometer length scale. Like the planar system, developing colloidal MEF systems include three main stages: i) Synthesis of metal core, ii) formation of spacer on metal core with sufficient thickness, and iii) Conjugation of biorecognition element to the as‐formed core/shell nanoparticles. However, stability of the colloidal nanoparticles system is a challenging issue that should be considered in the design of MEF biosensors because undesirable aggregation of metal NPs could alter the MEF response  dye which is a dsDNA intercalator.2, leading to a weaker fluorescence. Therefore, the fluorescence intensity of the biosensing probes is inversely proportional to the concentration of ATP in the solution with a wide dynamic detection range (100 nM–10 mM).The colloidal MEF biosensors can also be designed based on a competition assay. Figure 4.2.2.22+ ion, while its fluorescence could be recovered upon adding pyrophosphate due to the formation of pyrophosphate–Cu2+ complex. As pyrophosphate is a by‐product in the process of rolling circle amplification coupled with the ligase chain reaction, this Au nanocube@Silica@5‐carboxyfluorescein can be employed as the “turn‐on” probe to detect single nucleotide polymorphism . While the fluorescence of rhodamine B isothiocyanate (RITC) is quenched on the surface of individual Au@Ag nanoparticles, its fluorescence is “turned on” after forming plasmonic hot‐spots, allowing determination of the concentration of cysteine molecules in the colloidal MEF biosensor  has a wavelength different from the laser wavelength. This provides useful information about chemical structure, phase and polymorphy, crystallinity and molecular interactions of the studied molecule. This non‐destructive chemical analysis technique is called Raman spectroscopy. However, the Raman spectrum is usually weak at low concentrations of analyte, which restricts the use of Raman spectroscopy for biosensing applications when the analytes are usually low in quantities.4–108‐fold, which is highly sensitive to allow detection of biological analytes at ultralow concentration and even at the single molecular level. There are electromagnetic field and chemical enhancement mechanisms in SERS. The electromagnetic field enhancement refers to the enhanced excitation rate in the Raman probe as a result of the enhanced electric field around the metal nanostructure under the excitation laser. It has been reported that the excitation rate is proportional to the power of four of the enhanced electric field (E4E04) at the position of Raman probe. This factor enables the researchers to predict the best size, shape and even the percentage of surface coverage on metal NPs for the maximum SERS enhancement factor via computational simulations.Surface‐enhanced Raman scattering (SERS) is a physical phenomenon when the Raman scattering signal of a molecule is enhanced by the plasmonic nanoparticles. The typical enhancement factor for SERS is about 105.2Figure 5.2.12O2 induces deboronative hydroxylation of the Raman reporter , leading to a stable change in the SERS intensity between the two main peaks at 998 cm−1 and 1074 cm−1 of this molecule.In this review, we only focus on design of planar SERS biosensors based on the binding of colloidal nanoparticles onto the surface of substrate due to its simplicity and potential of large‐scale fabrication. The glass and silicon substrates are popular for developing planar SERS biosensors. The number of hotspots could be controlled by the number of metal NPs bound on the surface of substrate. Hamad‐Schifferli's group has immobilized the Zika and Dengue antibodies‐functionalized Au nanostars and Raman reporter on the surface of glass to interact with the viruses.Recently, planar SERS sensors using other substrates such as silicon (Si) wafer have been reported. Lin et al.5.2.2The colloidal SERS biosensors are ultrasensitive diagnostic tools that could be used for the detection and quantification of a wide range of analytes from ions and small molecules to bio‐macromolecules, viruses and pathogenic bacteria. Depending on the design of biosensor, the analyte could directly interact with the functionalized metal nanoparticle where the Raman spectra of the analyte can be measured and used for the quantification of the concentration of analyte in the sample. However, incorporation of a Raman reported molecule into the design  is necessary to correlate the existence of the analyte molecule to the Raman spectra of Raman reporter.5.2.2.12+ ions in aqueous solution. The strong coordination between the nitrogen atom of polyaniline and Hg2+ ions resulted in a lower stability of the designed Janus nanoparticle, leading to particle aggregation. The formation of hot‐spots between the Au@Ag nanorods as a result of aggregation enhances the Raman spectra of polyaniline, which was proportional to the concentration of Hg2+ ions  or a body‐fluid from human or animal . Ning et al.s Figure b.On the other hand, metal NPs functionalized with different recognition elements allows the simultaneous detection of multiple analytes, which approach is also known as the plasmonic nose. However, the analytes or the Raman reporters used in this approach should have a distinct Raman peak to better differentiate the molecules by SERS spectra. As shown in Figure 5.2.2.2Colloidal SERS biosensors are especially suitable to detect the biomarkers for in‐vivo diseases diagnosis, monitoring of specific biochemical phenomenon inside the cells as well as detecting live pathogenic bacteria. For example, gold nanostars have been used to study the interaction between avb3 integrin on human metastatic colon cancer cells and the arginine−glycine−aspartic acid (RGD) peptide. The Raman signal from the cysteine residue in the protein allows to monitor this integrin‐specific interaction via SERS.3O4 nanoparticles to detect three different pathogens . In this biosensor design, Ag@Fe3O4 NPs were functionalized with a pair of antibody and Raman reporter specific for each pathogen separately. The mixture of functionalized nanoparticles was then used to detect three kinds of pathogens simultaneously, achieving a wide range of linear response (10–107 CFU/mL) and low limit of detection (10 CFU/mL)  as the plasmon enhancer for SERS. The enhanced electric field of Au nanobones led to ultrasensitive SERS detection of Escherichia coli O157 : H7 with a LOD of 3 CFU/mL  Figure b. To furL Figure c.6Synthesis of anisotropic nanoparticles, especially gold and silver, has witnessed a huge progress within the last twenty years. Different methods have been developed to synthesize highly monodispersed nanoparticles with good control of morphology. As the optical properties of metal nanoparticles largely depend on their size and shape, it is important to develop reliable synthesis approach to produce a variety of metal NPs in high yield suitable for different applications. In particular, anisotropic metal NPs possess dramatic enhanced electric field at their sharp corners and edges under a wide range of excitation wavelengths, from visible to near‐infrared region, making them highly suitable to be used as plasmon enhancer in metal‐enhanced fluorescence (MEF) and surface‐enhanced Raman scattering (SERS). Among the optical biosensors, MEF and SERS stand out to be the most sensitive techniques to detect wider range of analytes from ions, biomolecules to macromolecules and even living microorganism. Besides the robust synthesis of anisotropic nanoparticles for plasmon enhancement, design of metal‐enhanced biosensors also depends on the selection or screening of specific biorecognition elements such as aptamers or antibodies for targeted bio‐detection. The right combinations of metal nanoparticles, biorecognition element as well as their functionalization and assembly will lead to successful designs of MEF and SERS biosensors.The applications of SERS and MEF biosensors have increased rapidly during last few years. However, the need for high‐tech instrumentations as well as highly diluted samples have hindered their progress toward clinical diagnostics. Some of these limitations could be addressed by developing brighter MEF and stronger SERS probes as well as different assay designs including the aggregation‐induced plasmonic hotspots formation through bio‐directed or self‐assembly. In addition, microfluidic and capillary  systems can be used to develop high quality metal nanoparticle for biosensor development with high reproducibility and reliability in detection. Despite the successful development of anisotropic nanoparticles of various shapes, limited number of these nanoparticles have been used in MEF and SERS biosensors. This shows a research gap to be addressed in the future, especially the development of next generation metal‐enhanced biosensors which require high selectivity, ultrasensitivity and great accuracy in the diagnostic results.cum plasmon‐enhanced nanosensing probes for theranostic treatment.For in‐vivo applications towards clinical diagnosis, MEF and SERS nanoprobes also restricted by the relevant issues such as strong background signals from the biological tissues as well as the penetration depth of laser beam to reach the targeted site  in living body. To address these issues, different groups of researchers have been working on the development of 1) new nanomaterials with suitable absorbance and emission in biological windows to minimize these background signals and 2) new generation of laser beams to have a deeper penetration in biological tissues. Besides Raman signal or fluorescence enhancement, metal nanostructures can also enhance other properties such as singlet oxygen generation (SOG) by placing the photosensitizer near to the metal NPs. This opens the door for the development of multifunctional nanomaterials with both diagnostic  and therapeutic functions  for image‐guided photodynamic therapy. Moreover, anisotropic gold nanostructures such as Au triangular nanoplates with flat surface (for better adsorption on tumors/cancer cells) and absorption in the NIR region can also be employed as the photothermal therapy agent We believe that the metal‐enhanced biosensors including MEF and SERS systems as reviewed herein possess promising potentials to be the new generation theranostic tools in nanomedicine. Its success for practical applications strongly depends on the accomplishment in the development of new theories, modelling and simulation of these processes as well as standardizing different procedures for the fabrication of substrates  and data analysis in the real and complex mediums, and finally, systematic studies for clinical translation and human testing.The authors declare no conflict of interest.Yen Nee Tan is an Associate Professor of Chemical Engineering at the Newcastle University. She obtained her PhD degrees from MIT and National University of Singapore under Singapore‐MIT Alliance Scholarship. She is also the Principal Investigator of Biosensors and Nanomaterials at the Newcastle Research & Innovation Institute (NewRIIS) in Singapore. She has authored over 50 journal publications, book/chapters and hold 18 patents on nanobiotechnologies for analytical and biomedical applications. Her current research focuses on the development of multifunctional bio‐hybrid nanomaterials and green technologies inspired by Nature for biomedicine, environmental detection and food security purposes.Mohammad Tavakkoli Yaraki received his Ph.D. in chemical and biomolecular engineering from National University of Singapore under the supervision of Prof Yen Nee Tan and Prof Bin Liu. His Ph.D. thesis was about the synthesis of metal nanoparticles and applications for metal‐enhanced singlet oxygen generation of photosensitizers with aggregation‐induced emission. The outcomes of his research have been published in several peer‐reviewed journals. His current research interest is design and development of hybrid nanomaterials containing plasmonic metal nanoparticles for various analytical purposes as well as biomedical applications.


Midazolam significantly reduced connectivity in the DMN . The effect sizes for ketamine for resting networks showed a positive correlation  with the effect sizes for schizophrenia‐related deficits derived from ENIGMA's study of 261 patients and 327 controls. Effect sizes for midazolam were not correlated with the schizophrenia pattern . The subtraction of ketamine and midazolam patterns showed a significant positive correlation with the pattern of schizophrenia deficits . RsPhfMRI reliably detected the shared and divergent pharmacological actions of ketamine and midazolam on cerebral networks. The pattern of disconnectivity produced by ketamine was positively correlated with the pattern of connectivity deficits observed in schizophrenia, suggesting a brain functional basis for previously poorly understood effects of the drug.Subanesthetic administration of ketamine is a pharmacological model to elicit positive and negative symptoms of psychosis in healthy volunteers. We used resting‐state pharmacological functional MRI (rsPhfMRI) to identify cerebral networks affected by ketamine and compared them to the functional connectivity (FC) in schizophrenia. Ketamine can produce sedation and we contrasted its effects with the effects of the anxiolytic drug midazolam. Thirty healthy male volunteers (age = 19–37 years) underwent a randomized, three‐way, cross‐over study consisting of three imaging sessions, with 48 hr between sessions. A session consisted of a control period followed by infusion of placebo or ketamine or midazolam. The ENIGMA rsfMRI pipeline was used to derive two long‐distance  and one local  FC measures. Ketamine induced significant reductions in the connectivity of the salience network (Cohen's  A placebo‐controlled, randomized, three‐way cross‐over design was used where participants were blind to the drug they were receiving. Each subject participated in all three sessions. Individual sessions were separated by 48 hr to allow for a wash‐out of drug effects. Each session collected resting‐state imaging data. Participants were instructed to have their eyes open and fixated on a small cross on a projection screen during infusion. Written informed consent was obtained from all participants and the local Ethics Committee approved the study. The data analyzed here have been previously reported in . For the resting‐state data reported here, 437 volumes were acquired .All experiments were conducted using a 3 T MRI scanner  equipped with a 20‐channel head coil. BOLD fMRI data were acquired using a T2*‐weighted echo‐planar imaging (EPI) sequence (repetition time (TR) 2,200 ms, echo time (TE) 27 ms, flip angle 79°, 30 interleaved 3‐mm slices, field of view 215 mm, voxel size 3 Drugs were administered to a subanesthetic level through an intravenous line controlled by an infusion pump , programmed by a supervising anesthesiologist, located in the MR control room. Ketamine was administered with a 0.25 mg/kg bolus dose, followed by a 0.25 mg/kg/hr infusion. Doses were similar to those used in previous literature , resulting in predrug and postdrug administration datasets and removal of the 2‐min bolus period. The ENIGMA resting‐state analysis pipeline implemented in the Analysis of Functional NeuroImages (AFNI) software Cox,  was usedResting‐state network templates were defined based on the probabilistic regions of interest (ROIs) from an independent components analysis of the BrainMap activation database and resting‐state fMRI dataset  was designed to investigate changes in local spontaneous brain activity by performing a nearest neighbor analysis of similarity of the BOLD time series and assigning a Kendall's coefficient of concordance (KCC) score /2 is the mean of the Ri's; K is the number of time series within a measured cluster , that is, one given voxel plus a number of its neighbors, and n is the number of ranks  the nearest neighboring nodes can be defined spatially, which usually reflects the anatomical, morphological, and geometric features in a local brain structure; (ii) the rank‐based computation is efficient in the time domain; and (iii) this score is robust against noise by integrating noise‐filtering operations across both the spatial domain and the temporal domain. Of note, the ReHo values of the voxels near the border of the brain mask need to be discussed with caution. The ReHo map, the collection of all voxels' KCC score of each voxel with  26 nearest neighboring voxels, for each subject each condition, was calculated using the MATLAB function “y_reho.m” available in the DPABI_V3.0_171210 package. The ReHo calculation was performed in three‐dimensional volumetric space, and still the calculation on the two‐dimensional cortical surface is preferred for the better representation of the neural neighborhood across the heavily folded cortex  and predrug (preinfusion) rsFC measures from both the seed‐based and dual regression analysis approach for the ketamine, midazolam, and placebo conditions. We compared KCC scores from each of the resting state template ROIs between postinfusion and preinfusion of the drugs and the placebo.Paired‐sample d‐values)—for ketamine, midazolam and the placebo—was calculated for each of the resting‐state functional connections; and for seed‐based and dual regression analysis approaches, separately. Similarly, Cohen's d‐values (postinfusion relative to preinfusion) were calculated using KCC scores for each of the resting state template ROIs.The difference between postinfusion and preinfusion measures , keeping the ROIs and the functional connections within the same hemisphere. The reasons behind this are that the FPN exhibited strong within‐hemisphere interactions but with distinct patterns in each hemisphere and preferentially coupled to the DMN and language‐related regions in the left hemisphere but to AttN in the right hemisphere  the rsFC measures derived from RSNs, and KCC scores from ReHo methods between the postinfusion minus preinfusion for the drugs and the postinfusion minus preinfusion for the placebo.To determine the drug impacts beyond the placebo effect, we compared  followed by the auditory network  and default mode network  followed by the left attention network  and the left frontoparietal network . Other connections showing significantly reduced FC strength under midazolam were between the left middle frontal area and the left superior parietal area ; and from the left inferior parietal lobule to the left inferior frontal gyrus . The only functional connection that showed a significant increase in FC was from the left motor cortex to right motor cortex in SMN (p < .05)  for ketamine, midazolam, and the placebo  were found between rsFC measures in the seed‐based and dual regression approaches with ReHo KCC scores  and AN connections (p < .01) with ketamine administration. Significant reductions in FC strengths for the DMN and left AttN (lAttN) connections (p < .01) with midazolam  whereas AN, SN, SMN, and VN ROIs showed no significant changes with midazolam  and SN (Cohen's d = 1.13) were roughly twice the significant patient‐control differences observed in rsFC measures for SN (Cohen's d = 0.43), and AN (Cohen's d = 0.53) in schizophrenia  on the DMN connectivity was not significant in the patient‐control differences in schizophrenia (Cohen's d = 0.24). The effect sizes of ketamine were found correlated  with the network‐based patient‐control differences in the FC in schizophrenia, but no association was found for midazolam  on activities of the DMN, SN, and AN were about twice those observed in the patient‐control differences in these networks in schizophrenia and the correlation between the patterns of affected networks approached significance , presumably by reducing the sedation effects that both drugs exerted through DMN. Overall, this study suggests that ketamine challenges can produce the pattern of functional disconnectivity that mimics that observed in schizophrenia with exception of sedative effects of ketamine that can be corrected by contrasting with the effects of the midazolam challenge.A single‐blind, randomized cross‐over approach was used to investigate the shared and distinct effects of ketamine and midazolam on the activity of brain functional networks and compare them with the pattern of cerebral disconnectivity observed in schizophrenia patients. The effects of the two drugs were readily detected by three independent resting‐state analysis approaches. Administration of a placebo produced no significant changes. Both drugs reduced the activity in the default mode network (DMN) that we attributed to sedation. The effect sizes of ketamine  and localized (ReHo) measures of connectivity. Overall, these results are consistent with findings from an fMRI study that used ketamine as one of the functional contrasts . We believe this to be a minor limitation because there were no significant sex differences in the schizophrenia pattern of cerebral disconnectivity in schizophrenia (Adhikari et al., 5In healthy male volunteers, both ketamine and midazolam led to significantly disrupted rsFC on the DMN but actions of two agents on other networks diverged. The effects of two agents were robustly detected by three rsFC measures and the effect sizes were consistent across three analysis approaches. Effects of ketamine were positively correlated with the pattern of disconnectivity observed in schizophrenia patients. This correlation became significant after accounting for sedation effects of ketamine by subtracting effects of the midazolam.The authors declare no conflict of interest. PT and NJ received a research grant from Biogen, Inc., for research unrelated to the topic of this manuscript.Appendix S1 Supporting InformationClick here for additional data file.


A number of feedbacks regulate the response of Arctic sea ice to local atmospheric warming. Using a realistic coupled ocean‐sea ice model and its adjoint, we isolate a mechanism by which significant ice growth at the end of the melt season may occur as a lagged response to Arctic atmospheric warming. A series of perturbation simulations informed by adjoint model‐derived sensitivity patterns reveal the enhanced ice growth to be accompanied by a reduction of snow thickness on the ice pack. Detailed analysis of ocean‐ice‐snow heat budgets confirms the essential role of the reduced snow thickness for persistence and delayed overshoot of ice growth. The underlying mechanism is a snow‐melt‐conductivity feedback, wherein atmosphere‐driven snow melt leads to a larger conductive ocean heat loss through the overlying ice layer. Our results highlight the need for accurate observations of snow thickness to constrain climate models and to initialize sea ice forecasts. Arctic sea ice sensitivity to atmospheric warming is computed using a numerical adjoint, revealing warming can lead to lagged sea ice growthSnow melt—in response to early warming—is the key preconditioner, enabling large conductive ocean heat loss to support lagged ice growth Our quantity of interest is the monthly mean sea ice volume  in the evolution of the heat budget for the ocean‐ice‐snow system. Time series of heat content change, integrated over the full depth of the relevant  component is shown in Figure The pattern of the imposed warm perturbation, Figures The net vertical heat flux , and  ocean‐atAn enhanced conductive heat flux  in snow and ice resistance at the start Figures 1 and 4b2To ascertain the key role of snow in sustained ice growth in response to atmospheric warming, we repeated the adjoint sensitivity calculations in another simulation where snow was removed from the initial conditions and precipitation was turned off at runtime to prevent new snow from forming. In this snow‐free Arctic experiment, the positive sensitivity of ice volume with respect to air temperature seen in WARM is absent (figure not shown). Atmospheric warming leads to enhanced sea ice melt at short lag. The sea ice slowly recovers through the following growth season, never overshooting its original (unperturbed) volume.4Using a coupled ocean‐sea ice GCM and its adjoint, we have investigated the space and time‐evolving sensitivity of total sea ice volume in the Arctic to changes in near‐surface atmospheric temperature. Counter‐intuitively, we find that near‐surface atmospheric warming can lead to lagged sea ice growth. Specifically, we identify extended regions where warming at the end of the melt season enhances subsequent ice growth.The responsible mechanism is a negative conductivity feedback, wherein enhanced summer melt preconditions the system for faster growth in the following winter via enhanced conductive heat loss. Previous studies have focused on the importance of early ice thinning in this feedback (i.e., the negative sea ice‐conductivity feedback, Bitz & Roe, Unambiguous detection of negative snow‐melt‐conductivity feedback is enabled through calculation of time and space‐varying ice volume sensitivities to surface air temperature. To our knowledge, this is the first published adjoint‐based investigation suggesting that warming can lead to sea ice growth. We note this feedback is not evident in the adjoint‐based assessment of Arctic ice volume change by Koldunov et al. , who appWe have focused on the response of Arctic sea ice volume to atmospheric warming. We did not address the response of Arctic ice volume to near‐surface ocean warming, although this has received significant attention in the literature in recent years, amid growing evidence of enhanced upper ocean radiative heating of the Arctic Ocean (Moore et al., To elucidate the importance of the insulating snow layer, we imposed an artificial surface warming perturbation Figure , purposeSnow thickness at the beginning of the sea ice growth season determines the strength of the subsequent conductivity feedback described in this study. In turn, snow thickness depends on a variety of factors (see Figure 1 Webster et al., Supporting Information S1Click here for additional data file.Data Set S1Click here for additional data file.Data Set S2Click here for additional data file.Data Set S3Click here for additional data file.Data Set S4Click here for additional data file.