Cow’s milk protein allergy is one of the most common pediatric food allergies. It poses a significant socioeconomic burden in industrialized countries and has a profound effect on the quality of life of affected individuals and their families. Diverse immunologic pathways can lead to the clinical symptoms of cow’s milk protein allergy; some of the pathomechanisms are known in detail, but others need further elucidation. A comprehensive understanding of the development of food allergies and the features of oral tolerance could have the potential to unlock more precise diagnostic tools and novel therapeutic approaches for patients with cow’s milk protein allergy. Cow’s milk protein allergy (CMPA) poses a constant challenge for healthcare providers and patients alike. The increasing incidence of food allergies in industrialized, urban environments, the diagnostic difficulties physicians may face, the cost associated with the morbidity , and theCow’s milk allergy is a reproducible hypersensitivity reaction, consistently leading to a wide variety of symptoms, including gastrointestinal, respiratory, and skin manifestations, after the ingestion of the culprit food.The prevalence of the condition among infants is 2–6% and decreases with age, although the onset of symptoms may occur at any age . ApproxiThe epidemiology of non-IgE-mediated CMPA has been subject to fewer studies. Some national cohorts are helpful in estimating the prevalence of different conditions. FPIAP, as the most common disorder with non-IgE-mediated background, has been associated with a wide prevalence range of 0.16 to 7%, and the cumulative incidence is described to be around 17%. FPE shows a progressively decreasing incidence over time, and some studies indicate a prevalence rate of around 2%. FPIES is considered to be a rare condition; the cumulative incidence is estimated to be 0.3–0.7% in infancy [The aim of this review is to summarize the immunologic background of CMPA, the mechanism of oral tolerance, and possible treatment strategies already in use or under investigation.The two main subtypes of CMPA, based on the immunologic response to the allergen and the subsequent occurrence of symptoms, are IgE- and non-IgE-mediated allergies, although a mixed presentation caused by activation of both immunologic pathways also exists ,8 Table.Patients with IgE-mediated allergy show symptoms within minutes up to 2 h of culprit food ingestion; the severity ranges from relatively mild manifestations to life-threatening anaphylaxis, whereas non-IgE-mediated patients’ symptoms develop after a delay, usually within hours or days, rarely weeks.IgE-mediated CMPA can affect many different organ systems, mostly the skin and the gastrointestinal and respiratory tracts; the most frequently observed symptoms associated with this condition are acute urticaria, angioedema, pruritus, oral tingling, oral pruritus, swelling, nausea and/or vomiting, abdominal pain, wheezing, and systemic symptoms, such as hypotension, hypothermia, and potentially fatal anaphylaxis ,10.The clinical manifestations of non-IgE-mediated CMPA can be described as three clinical disorders: food protein-induced enterocolitis syndrome (FPIES), food protein-induced enteropathy (FPE), and food protein-induced allergic proctocolitis (FPIAP); an overlap between clinical symptoms is common. The severity of these manifestations represents a spectrum, with FPIAP being the most benign and FPIES usually being the most severe .The leading symptom of FPIES is profuse and repetitive vomiting, which can be followed by diarrhea in up to half of the patients. Lethargy, pallor, and hypothermia may also occur; rarely, hemodynamic instability develops in the affected patients .The clinical presentation of FPE is similar to celiac disease: after the introduction of cow’s milk to the infant’s diet, chronic diarrhea and malabsorption with indicative signs, such as steatorrhea and failure to thrive, develop. In contrast to FPIES and FPIAP, FPE may require endoscopy and histologic analysis to confirm the diagnosis .FPIAP usually develops within the first weeks of life of breastfed infants; they are exposed to cow’s milk protein through the maternal intake. Loose, bloody stools, in some instances with mucus, show inflammatory changes of the rectum and colon in otherwise healthy-appearing infants ,12.Eosinophilic gastrointestinal disorders  represent a distinctive group of illnesses among cow’s milk protein allergy manifestations. The exact pathophysiology of eosinophilic gastrointestinal disorders (EGIDs) is still under investigation, but they are typically categorized as “mixed” IgE and non-IgE mediated because allergic sensitization is often observed . ClinicaThe total protein composition of cow’s milk can be divided into two main fractions, which are obtained through the acidification of raw milk (pH 4.6 at 20 °C): the coagulum containing casein proteins  and the lactoserum . The casein fraction contains αs1-, αs2-, β-, and κ-caseins and three γ-caseins derived from the hydrolysis of β-casein (the latter being mostly abundant in cheeses rather than milk). Caseins are resistant to heat but susceptible to enzymatic degradation. One of the most important components of lactoserum is β-lactoglobulin, which contains linear IgE binding epitopes. Other components include α-lactalbumin  and bovine serum albumin ; finally, the allergenic activity and clinical significance of some components, such as immunoglobulin G and lactoferrin, require further investigation ,16 The mPatients with IgE antibodies against casein were found to have a higher likelihood of allergy persistence .Heating is a fundamental step for enabling the commercial availability of milk because of the reduction or elimination of microorganisms; during pasteurization, temperatures below boiling point are applied, whereas ultra-high-temperature processing uses temperatures exceeding 100 °C. Powdered, milk-based infant formula is produced through evaporation, during which exposure to high temperatures is usually short. However, heating seems to have a moderate effect on the allergenic potential of milk proteins, though some authors found a reduction in whey protein allergenicity. High temperatures can only modify conformational epitopes, whereas linear epitopes remain unaffected; hence they are still able to maintain their allergenic potential.Hydrolysis is utilized to alter the allergenicity of milk proteins, but only extensively hydrolyzed formulas are considered safe for patients with CMPA . A combination of ultrafiltration (which removes any undigested proteins remaining in the filtrate after proteolysis) and hydrolysis can further improve the safety of hypoallergenic products .Although some patients are able to tolerate baked milk, as a higher number of Treg cells and lower milk-specific IgE levels were detected in this patient population ,18,19, aThe key feature in food allergy pathogenesis is the lack of oral tolerance–a normal state of unresponsiveness–to food allergens. Sensitization is the preceding step in the development of symptomatic food allergy, and the “dual allergen exposure hypothesis” sheds light on multiple important mechanisms leading up to the development of food allergy. As opposed to the previous theory about sensitization, which emphasized the role of intestinal exposure  to dietary antigens, increasing evidence indicates an important role of early life allergen exposure through the skin in the subsequent manifestation of food allergy, whereas early oral exposure can establish tolerance ,22. The Food antigens are digested into peptides and amino acids; however, a small fraction can reach the intestinal epithelium in an intact form, which enables interaction with antigen-presenting cells.Dietary antigens are able to pass through the intestinal epithelium, depending on size and solubility, to subsequently access antigen-presenting cells in the mucosa via passive or active transport. Passive transport, also known as paracellular diffusion, happens when a food antigen passes between two adjoining enterocytes.Under homeostatic conditions, tight junctions between enterocytes prevent the paracellular passing of antigens. Increased transport of intact antigen through epithelial cells has been related to its allergenic activity.The active system takes place through different cell types. Microfold (M) cells are flattened epithelial cells overlying Peyer’s patches; sampling through these cells is associated with the induction of IgA production, which plays a critical role in the intestinal lumen. Goblet cells also take part in soluble antigen transport by creating goblet-cell-associated antigen passages (GAPs), which then deliver antigens to CD103+ CX3CR1- dendritic cells, a DC subset associated with the development of tolerance. Mucin secretion increases antigen transport through goblet cells and uptake by the aforementioned DC subset ; additioSpecialized macrophages with CX3C receptor 1 expression are able to pass captured antigen via trans-epithelial dendrites or phagocytosis onto DC for transport to mesenteric lymph nodes (MLNs) to prime immune responders, such as lamina propria DCs . CD103+ TGFß-expressing Th3 cells are induced after ingestion of antigen which shows some overlapping functions and possible plasticity with Foxp3+ T cells. Tr1 cells have immunosuppressive effects through IL-10 and, thus, are capable of preventing colitis, although their exact role in oral tolerance could not yet be determined because of conflicting research results .The transport of antigens to secondary lymphoid tissues, such as mesenteric lymph nodes or Peyer’s patches, promotes tolerance, although the exact importance of these secondary lymphoid organs needs further investigation ,31,32.A very small fraction of proteins (an estimated 2%) passes through the intestinal epithelium in intact form, which then could be transported to the liver or secondary lymphoid tissues for antigen presentation . After aMultiple Treg populations play a role in the development of oral tolerance, especially antigen-specific CD4 + CD25 + Foxp3 + Tregs, which is the most widely studied and understood group of Treg subsets, with a crucial part in the intestinal tolerance .Another factor in the complex development of oral tolerance is homing of Treg cells to the gut, which is promoted by the production of retinoic acid by CD103+ DCs and the subsequent expression of integrin α4ß7. A further human study that underlines the quintessential role of Treg cells showed that hypomethylation of the Foxp3 locus leads to its increased accessibility for transcription in patients who respond favorably to oral immunotherapy  .A potential capacity of tonsils in the development of oral tolerance has been described. The anatomical location and abundance of CD4+ Foxp3+ Tregs and allergen-specific Tregs indicate that these organs may represent the first phase of oral tolerance induction to food allergens .Besides the induction of regulatory Treg cells, anergy (unresponsiveness to the antigen) and T-cell depletion (apoptosis of antigen-specific T cells) are also mechanisms through which oral tolerance can be achieved. The former phenomenon occurs after high-dose antigen exposure, while the latter follows low-dose exposure .In patients with IgE-mediated food CMPA, the physiologic tolerance is disrupted, for example, after exposure to pathogen-associated molecular patterns (PAMPs) or epithelial damage, which leads to IL-25, IL-33, and thymic stromal lymphopoietin (TSLP) production. These changes cause an alteration in the induction of Treg cells, which are subsequently switched to antigen-specific Th2 cells. Th2 cells stimulate B cells through IL-4 production, which then results in immunoglobulin E (IgE) production and mast cell expansion. Tolerogenic Treg function is suppressed by IL-4, and finally, these cells are reprogrammed to produce IL-4 themselves, transforming from tolerogenic to pathogenic ones .Type 2 innate lymphoid cells (Th2-like cells without antigenic specificity), which secrete IL-4 and IL-13, further inhibit Treg activity . SecreteThe molecular background of non-IgE-mediated CMPA is understood in far less detail than the IgE-mediated variant. Cellular immunity is presumed to be the key factor in the allergic response in the absence of circulating sIgE, although localized intestinal IgE response has been previously described .Research indicates the presence of food-specific T cells in FPIES, but their distinctive role is yet to be identified. Although Th2 responses are usually described in association with IgE-mediated allergy, their apparent role in FPIES  can be explained by the high rate of atopy as a comorbidity in these patients . StudiesStructural damage to the jejunal mucosa and subsequent malabsorption are important features of FPE, which seem to be caused by food-specific T cells (predominantly cytotoxic CD8 + T cells) infiltration ,42.Dense eosinophilic infiltration of the rectosigmoid mucosa is characteristic of FPIAP. Since breastfed infants are predominantly affected by the disease, a potential role of immunologic components in breast milk and their interaction with dietary proteins has been proposed .Eosinophil gastrointestinal disorders are characterized by abnormal eosinophilic infiltration of the esophagus, stomach, small intestine, or colon, leading to organ dysfunction and clinical symptoms. Although the pathomechanism of these conditions still needs further clarification (eosinophilic esophagitis is the most studied subtype of these disorders), available data indicates an allergic background  of mixed immunologic mechanisms (IgE- and non-IgE-mediated) .The central role of the Th2 response in the pathogenesis of eosinophilic esophagitis, with the release of mediators such as thymic stromal lymphopoietin (TSLP), IL-4, IL-5, IL-13, TGFß, and eosinophil chemokines (eotaxin 1-3/CCL11-CCL24-CCL26 and RANTES/CCL5) has been demonstrated . A lowerCMPA has been linked to a variety of nonspecific symptoms, such as allergic dysmotility , without a complete understanding of the underlying pathomechanisms. Animal models indicate a significant modulating role of allergic responses on intestinal motility. A Th2-dominant response to food allergens causes the release of IL-4 and IL-13, which alter smooth muscle motility and spontaneous contractility via the TGFß upregulation . AnotherThe initial steps toward the diagnosis of CMPA are a thorough clinical history (preferably guided by open-ended questions) and a physical examination. If an IgE-mediated allergy is suspected, measurement of specific serum IgE levels (sIgE) and a skin prick test (SPT) are a reasonable next step, though it should be noted that these tests alone are insufficient for establishing the diagnosis of CMPA ,48. SPT has a high negative predictive value, but a positive test is not suitable for confirming the diagnosis of CMPA; it rather indicates a state of sensitization. An allergen extract is transferred to the tip of a small lancet, which penetrates the epithelial barrier, causing mast cell degranulation in susceptible individuals, which presents as a wheal-and-flare reaction .The main advantage of measuring specific serum IgE levels is the correlation of s-IgE concentrations with the possibility and severity of a clinical reaction to the allergen, although an exact cutoff value is difficult to determine  .Before the subsequent OFC, which is still the gold standard for establishing the diagnosis of food allergy, the aforementioned diagnostic methods are appropriate for risk assessment.During OFC, increasing doses of the suspected allergen are administered orally until either clinical symptoms develop or a maximum tolerated dose is reached .It is ideal for conducting it in a double-blind, placebo-controlled (DBPCFC) design, but due to the high costs and time commitment required, it is rarely used in the clinical setting. In addition, oral food challenges should take place in specialized hospital wards with personnel and equipment suitable for promptly managing acute allergic reactions.Fecal calprotectin has been successfully utilized for the diagnosis and monitoring of gastrointestinal inflammation over the last decades because the quantification of this biomarker is a simple, fast, and relatively inexpensive procedure. It may be a valuable tool, as some studies have recently demonstrated, in the medical diagnosis of the non-IgE-mediated CMPA .A promising direction toward a more precise and objective diagnosis of food allergies is component-resolved allergy testing. In this method, purified or recombinant allergens are used to identify allergen-specific IgE and IgG4 antibodies . A signiThe basophil activation test (BAT) is a promising, safe in vitro diagnostic method; although it is mostly used in research settings, it could provide a highly accurate diagnosis of IgE-mediated food allergies. During BAT, the activation of basophils via the IgE receptor leads to an increase in surface markers (CD63 and CD203c), whose level of expression is measured via the flow cytometry ,38.The most widely accepted and utilized concept for the management of cow’s milk protein allergy is a “passive” approach, which means the complete elimination of cow’s milk and any dairy from the patient’s diet. While a high adherence to this diet leads to the resolution of symptoms in most cases, the risk of potentially life-threatening reactions and a possible detrimental impact on the nutritional status and overall quality of life of affected individuals warrant the continuous investigation of new, “proactive” therapeutic options. In the infant population, breastfeeding, if desired by the mother, can usually be continued if she is able to follow a strict dairy-free diet, thus eliminating the infant’s exposure to the allergen through breast milk. A switch to an extensively hydrolyzed formula (EHF) or, in the most severe cases of CMPA, amino-acid-based formula (AAF) is recommended ,10. ThroIn older children, a standard elimination diet and, in the case of involuntary allergen intake in severe IgE-mediated cases, carrying an adrenaline autoinjector are required to maintain remission and effectively mitigate life-threatening reactions; however, the focus is shifting toward therapeutic options that could possibly help to establish a state of sustained unresponsiveness or even permanent tolerance.Immunotherapy for food allergies can be classified based on the route the allergen is administered: oral immunotherapy (OIT) requires oral ingestion of the allergen; sublingual immunotherapy (SLIT) is characterized by holding the allergen under the tongue for 2 min; and epicutaneous immunotherapy (EPIT) relies on applying an allergen patch to the skin ,55 Tabl.A conventional OIT protocol consists of three phases. During the initial escalation phase, which is carried out under supervision in a healthcare facility, the aim is to determine the starting dose (the highest safely tolerated dose) for daily administration at the patient’s home. This step is followed by a buildup phase, during which the daily dose is gradually increased at regular intervals  until a maintenance dose is reached. Finally, this maintenance dose of the allergen should be consumed by the patient daily for an extended period (months or years). After the maintenance period, the daily ingestion is suspended for 4 to 12 weeks to assess sustained unresponsiveness with DBPCFC. A lack of a clinical reaction is indicative of a permanent tolerance ,56.The immunologic background of the mechanism of OIT remains unclear. It is proposed that frequent allergic stimulation leads to mast cell desensitization and the induction of allergen-specific Foxp3+ Tregs. Continuous high-dose allergen stimulation may induce IgG subclass switching (μ → γ3 → γ1 → γ2 → γ4) rather than sequential class switching . Allergen-specific IgG4 antibodies are able to compete for allergen binding, thus inhibiting mast cell and basophil degranulation. IgG antibodies induced during OIT could act through the inhibitory receptor FcγIIb to decrease IgE-mediated hypersensitivity. In the early stages of OIT, dendritic cells produce IL-10, interferon- γ, and decreased levels of IL-6, which suppress the IgE-mediated hypersensitivity .Combining OIT with omalizumab, an anti-IgE monoclonal antibody is studied as a possible augmentation of OIT efficacy through alleviating the severity of adverse reactions and facilitating a faster achievement of a maintenance dose ,57,58. ASLIT and EPIT, although associated with fewer adverse reactions and better patient adherence, have scarcely been studied in patients with cow’s milk protein allergy; therefore, robust conclusions about their efficacy cannot be drawn ,63,64. ANon-IgE-mediated CMPA has a more favorable prognosis, although the quickest possible achievement of oral tolerance is a reasonable objective in the therapeutic approach. Some studies suggest the beneficial role of probiotics (besides the elimination diet) in tolerance acquisition ,67,68,69A continuous increase in the number of CMPA patients means a growing burden on the healthcare sector, children, and their families, which accentuates the importance of ongoing research in this field. A clearer insight into the pathomechanism of CMPA on a molecular level may facilitate the recognition of new potential therapeutic targets in the future.While the molecular features of IgE-mediated allergies have been described in significant detail, some mechanisms of non-IgE-mediated allergies and mixed-background allergic diseases still remain enigmatic and need further research and understanding in order to explore a broader spectrum of diagnostic and therapeutic opportunities.A paradigm shift (from “passive” to “proactive” management) is shaping the therapeutic approach to CMPA, which highlights the need for a thorough comprehension of the diverse immunologic mechanisms associated with cow’s milk ingestion in allergic individuals. Comprehensive knowledge about the mechanisms of the development of cow’s milk protein allergy can also further enhance current prevention strategies.


D1 inhibition, which seems to be related to the presynaptic inhibition, was recorded by measuring soleus Hoffmann’s reflex (H-reflex) with conditioned electric stimuli to the common peroneal nerve. Results: D1 inhibition was significantly enhanced immediately by the cycling intervention. The amplitude of the soleus Hmax was significantly depressed, and there was significant difference in Hmax/maximum M-wave. The MAV was increased due to inhibition of the stretch reflex. Conclusion: Leg cycling suppressed stretch reflex and H-reflex, and caused plasticity of inhibitory circuits in patients with CP with lower extremity spasticity. These findings strongly suggest that lower extremity spasticity can be improved by cycling movements.Objective: The purpose of this study was to investigate if leg cycling could reduce lower extremity spasticity in patients with cerebral palsy (CP). In addition, we investigated whether the intervention could cause changes in the modulation of presynaptic inhibition. Methods: This study was a quasi-experimental study, with pretest–posttest for 1 group. Participants in this experiment were eight adult patients with CP with lower extremity spasticity. Spasticity parameters assessed were the amplitude of soleus maximum Hoffmann’s reflex (H Cerebral palsy (CP) includes a group of permanent disorders of movement and postural development that cause activity limitations and can be attributed to non-progressive disturbances in the developing fetal or infant brain1). The motor disorders associated with CP are often accompanied by secondary musculoskeletal problems2). Spasticity is defined as a motor disorder characterized by velocity-dependent stretch reflexes with exaggerated tendon jerks, which is a symptom of upper motor neuron syndrome3). Spastic CP accounts for approximately 80%–90% of all patients with CP4); they suffer from symptoms such as muscle stiffness5), shortening, and increase risk for falls6). Moreover, the contracture responsible for stiffness causes further muscle overactivity, including spasticity7). Although spasticity has been attributed to lesions of the pyramidal tract, in animals, isolated lesions of the primary motor cortex (Broadman area 4) have been shown to decrease muscle tone and tendon reflexes instead of causing spasticity. Moreover, lesions of the premotor cortex and supplementary motor cortex (Broadman area 6) have been often shown to induce spasticity8,9). Even though pathophysiological mechanisms underlying spasticity in humans are not completely understood, the evidence suggests that an indirect descending pathway modulating the stretch reflex circuit from the motor cortex, such as the vestibulospinal tract, and presynaptic Ia inhibition is one of the spinal inhibitory interneuron systems involved in stretch reflex modulation10,11). D1 inhibition12), which seems to be related to the excitability of primary afferent depolarization interneurons mediating presynaptic inhibition of Ia terminals, deteriorates in CP13). In addition, several studies have reported that patients with stroke14) and multiple sclerosis15) show increased Hoffmann’s reflex (H-reflex) amplitude and impaired presynaptic Ia inhibition in the lower limbs in comparison with healthy controls.16), while no established evidence could be identified for therapeutic exercise. Nevertheless, we have experienced decreased muscle tone in patients with CP after cycling exercise in our daily clinical practice. Recent investigations have demonstrated that cycling movements induce a reduction in spasticity variables such as the H-reflex amplitude and Modified Ashworth Scale (MAS) scores17) and enhance inhibitory spinal pathways such as those mediating presynaptic inhibition in patients with diseases of the central nervous system other than CP18–21). In contrast, only a few studies have focused on the combination of electrophysiological measurements and use of this therapeutic intervention for spasticity in CP, and its effects on the modulation of spinal inhibitory interneuron systems in patients with spastic CP.Considering these findings, appropriate interventions for spasticity are extremely important, and its management is currently achieved through various therapeutic exercises, pharmacotherapy, and orthopedic surgery. In a recent systematic review on the evidence for interventions for CP, botulinum toxin, diazepam, intrathecal baclofen, and selective dorsal rhizotomy were recommended for reducing muscle spasticity22,23). The common core hypothesis suggests the existence of central pattern generators (CPGs) and regulatory systems that modulate reflex output within the spinal cord. The reason for focusing on D1 inhibition is that presynaptic inhibition may contribute to inputs from higher centers, CPGs, and even peripheral sensory receptors to modulate reflex outputs in the spinal cord.Rhythmic movements such as cycling and walking have different neural controls than normal voluntary movements24–26), and little evidence is available for the quantitative evaluation of spasticity. In this regard, the pendulum test27), which is performed by freely dropping the lower leg from knee extension in a relaxed state, has proven to be a reliable and objective method to assess various aspects of spasticity in CP28,29).Moreover, the spasticity in CP was often evaluated using the MAS according to published literature and in clinical practice, although some studies have questioned the reliability and validity of this evaluationThis study aimed to investigate if leg cycling could reduce lower extremity spasticity in patients with CP. In addition, we investigated whether the modulation of presynaptic inhibition  changed after this intervention.This study was a non-randomized, quasi-experimental study, with pretest–posttest for 1 group.30) (GMFCS). Six patients with CP were classified as level I and two were classified as level III. CP subtypes were also assigned by classifying four patients with spastic diplegia, one with hemiplegia, and three with mixed CP. While kinematic measurement was feasible for all participants, electrophysiological measurements showed that so while D1 inhibition was evaluable in seven patients, the maximum M wave (Mmax) in the soleus muscle was obtained from five participants because three of them could not tolerate the maximal supra-stimulus intensity to the nerve. The study protocol was approved by Sapporo Medical University Hospital Institutional Review Board (Permit Number: 282-32) and was conducted in accordance with the Declaration of Helsinki of 1975, as revised in 2013. Written informed consent was obtained from patients who participated in this study.Fourteen adult patients with CP were recruited from Sapporo Medical University Hospital. Six of them were unable to start the electrophysiological measurement protocol because of discomfort and excessive involuntary movements in response to the slight stimulus. The remaining eight patients  finally participated in this study. The functional level of participants was classified according to the Gross Motor Function Classification SystemInclusion criteria were: (1) adults with CP and lower extremity spasticity (quadriceps and soleus muscle MAS score ≥1), (2) ability to comply with simple verbal directions, (3) GMFCS levels I to III, (4) ability to pedal the ergometer, and (5) knee extension range of motion ≥–30°.Exclusion criteria were: (1) orthopedic surgery within six months preceding the experiment, (2) a history of botulinum injections or other medications used in the treatment of spasticity within three months preceding the experiment, and (3) difficulty in cycling without orthoses.Electrophysiological and kinematic measurements were obtained using the H-reflex method and pendulum test, respectively. Each measurement was obtained only for the more affected spastic lower extremity.The participants were required to take a break of 10 min before the experiment, subsequently seated comfortably in a wheelchair with the knees flexed approximately 70° and the ankle joint fixed in the mid position. Electromyographic (EMG) activities were recorded using bipolar surface electrodes  placed 2 cm apart over the muscle bellies of the soleus and tibialis anterior .12).The EMG signals were amplified and sampled at 2000 Hz, and band-pass filtered at 10–1000 Hz, capable of making measurements across a ±5 mV range (10 mV span) using an 16-bit A/D converter connected to a computer . Soleus H-reflexes were elicited by percutaneous stimulation  of the posterior tibial nerve in the popliteal fossa through the bipolar stimulating electrode. D1 inhibition was evoked by soleus H-reflex induced with an electrical stimulus applied to the common peroneal nerve (CPN)max), and both conditioned and control H-reflexes were recorded in 10 sweeps each (max to Mmax (Hmax/Mmax) was determined in the recruitment curve of each participant.The conditioning stimulus was generated at the dorsolateral aspect of the caput fibulae with a rectangular pulse of 1 ms duration and an intensity that was 1.2 fold the motor threshold, and it preceded the test stimulus with a 20-ms interstimulus interval (ISI). The control H-reflex intensity was adjusted at 50% of the ipsilateral soleus maximum H-reflex  was attached to the front of lower leg distal to the side tested, and the swinging motion with angular velocity of the rotation around the X axis was recorded and sampled at 250 Hz. The examiner passively lifted the lower limb to full extension and held the leg position until the state of complete relaxation for 5 s or more, as indicated by palpation and inspection of quadriceps contraction and spasm. Subsequently, the relaxed limb was released to swing freely and allowed to oscillate until it stopped at a resting position. The maximum angular velocity (MAV) during the first swing, which was obtained using the gyro sensor, was stored on a computer for subsequent offline analysis. As for data processing, zero point correction was performed on angular velocity from data in which the sensor was stationary for 3 s for each patient.All participants performed a 20-min exercise at a 20-W constant work rate on the ergometer. Adjustable foot straps and magic tape were used to hold the participants’ feet and prevent slipping. The saddle height was adjusted so that the knee flexion angle was approximately 20°–30° at the bottom dead center of the pedal. The participants were instructed to cycle at a comfortable rate with smooth movements without clonus and stretch reflexes. Therefore, we did not control the rotation speed for each participant, although made sure that it was not less than approximately 20 rpm. The positions of the surface electrodes and accelerometer were ensured to remain constant during the intervention.31) for only parametric tests . Statistical significance was determined by a p-value of 0.05.Statistical analyses were performed using the Statistical Package for the Social Sciences version 21 . The results were expressed as mean ± standard error of the mean. Differences between the measurements before and after the intervention were calculated with a paired two-tailed t-test. The effect size was calculated by Cohen’s dmax were significantly depressed , but no significant differences were observed in Mmax before and after the intervention , and so there was a significant reduction in Hmax/Mmax ratio . In addition, the MAV values significantly increased after the intervention  enhancement of D1 inhibition, (ii) suppression of H-reflex amplitude, and (iii) increase in MAV.10). An increase in D1 inhibition reflects the enhancement of presynaptic Ia inhibition, which is one of the inhibitory mechanisms in spinal neural circuits that are impaired in patients with spasticity13–15). In a previous study, the mean suppression of the H-reflex by D1 inhibition was 86.19% on the paralyzed side and 77.64% on the non- paralyzed side in stroke patients with spasticity (n = 29)13), 87.56% in children with CP (n = 21), and 69.56% (n = 21) in non-disabled children14). These findings are similar to the pre- and post-intervention suppression in the present study. Long-term cycling exercises in patients with spinocerebellar ataxia causing coordination defects have been shown to enhance D1 inhibition concurrently with improvements in ataxia score32). Thus, the enhancement of D1 inhibition may be related to agonist and antagonist control in the cycling task. Spastic CP is characterized by impairments in coordination and selective motor control in the lower extremities, particularly the distal joints1,33). Patients with spastic CP show high co-contraction ratios for the lower limb muscles during a single cycling session compared with healthy participants34). Whether an improvement in selective motor control of the lower extremities leads to enhanced D1 inhibition is a topic for future studies.The currently accepted hypothesis of spasticity focuses on inhibitory rather than excitatory mechanisms in the spinal circuit18,19,35,36). Furthermore, other studies have reported that the soleus H-reflex decreases after arm cycling exercise19). When it was conditioned with CPN stimulation during arm cycling37), a greater reduction than that in static control was observed. These findings support the role of excitability of presynaptic inhibition in the activation of supraspinal and propriospinal pathways and the CPGs in cyclic arm movement. Neural mechanisms such as the CPGs and peripheral feedback during rhythmic movement have specific modulatory effects on reflex inhibition38). The increased presynaptic inhibition in cycling probably contributed to the degradation of the input of Ia afferent fibers and suppressed the soleus H-reflex; additionally, D1 inhibition was enhanced in this study. Previous studies have shown that the soleus H-reflex is depressed by continuous passive and active cycling for at least 30 min. Since passive cycling involves almost no muscle contraction, afferent input from types III and IV afferent mechanoreceptors may be associated with presynaptic inhibition of Ia afferent terminals and contributes to reflex control35,39).Another possibility is that increases in presynaptic inhibition by rhythmic and reciprocal movements influence several spinal pathways and afferent fibers. Numerous studies have demonstrated that the H-reflex of the lower limb changes during and after lower extremity cycling exercise in a task-dependent manner40). Post-activation depression41), one of the presynaptic mechanisms underlying spasticity, is related to muscle spasticity13,14). However, a previous study has reported that inhibition of spasticity is not explained by its depression since cycling training decreased the soleus H-reflex and MAS score for calf muscle in contrast to the sustained post-activation depression observed in patients with multiple sclerosis19). Thus, H-reflex suppression after cycling exercise in patients with CP may have been caused by the modulation of presynaptic inhibition rather than post-activation depression.These findings suggest that downregulation of the H-reflex is less related to descending motor pathways such as corticospinal or corticobulbar tracts and is more associated with increased fusimotor drive, Ia activity, and primary afferent depolarization28). Another study found that among 20 children with CP, the mean MAV improved from 244°/s to 364°/s after selective dorsal rhizotomy to control spasticity29). In our study, the increase in the MAV was statistically significant, although it was not close to this value.The increased MAV indicated that the velocity-dependent stretch reflex in the quadriceps was inhibited by this intervention. The MAV (mean ± standard deviation) for children with (n = 10) and without (n = 10) CP in a previous report was 201.82 ± 67.96°/s and 292.51 ± 35.93°/s, respectively, suggesting that MAV is significantly lower in children with CP35,36). In the present study, the stretch reflex of the quadriceps was suppressed. While there are no studies assessing quadriceps stretch reflexes after pedaling, studies report decreased hamstring MAS42). Since pedaling generates muscle activity in the quadriceps, hamstrings, and lower leg in CP34), it is possible that the suppression of the reflex occurred in a task-dependent manner not only in the soleus muscle but also in the quadriceps.Suppression of the soleus H-reflex and enhancement of D1 inhibition may be the result of a specific effect of pedaling exercise. Previous studies have described task-dependent changes in the soleus H-reflex during and after pedaling in able-bodied people13) . In contrast, another study found that spastic stroke patients have impaired D1 inhibition compared to healthy participants14). Regarding the amount of H-reflex suppression, it is possible that stroke patients have a smaller degree of D1 inhibition of the damage despite the shorter time from onset than hemiplegic CP.The suppressed H-reflex in D1 inhibition could not be confirmed in the hemiplegic patient . Strongly impaired D1 inhibition in spastic hemiplegic CP has been previously reported, but the number of participants was small43,44). In the participants with hemiplegia in our study, D1 inhibition was not modified, whereas the reflex  was inhibited. Hence, the exaggerated reflex in hemiplegic CP must be affected by mechanisms other than presynaptic inhibition, but further data accumulation is necessary.We hypothesized that impaired H-reflex inhibition in hemiplegic patients with CP may be related to the timing of brain damage in infancy or fetal life because GABAergic neurons in the central nervous system have been shown to change from excitatory to inhibitory synapses during early neuronal development in animal testingThe clinical significance of these results is that the enhancement of the spinal inhibitory interneuron is a result of reciprocal movement. Moreover, the cycling task is easy to use in clinical practice and can be recommended as an exercise therapy. For future studies, the effect on motor functions such as walking speed and endurance, as well as the long-term effects and required duration of intervention need to be further examined. The intervention time in this study was within the same time frame as that of several previous studies, and similar results were obtained. However, there are reports of changes in spasticity scores in even shorter time periods.13,14,32). However, it is important to note that this value may vary among individuals12). Moreover, this experiment was performed with participants showing several types of CP , a wide range of ages, and a relatively small sample size, which could limit the interpretation of the results. Nevertheless, we anticipate that cycling movements will become an effective intervention for the treatment of spasticity in patients with CP.The present study has some limitations. The ISI required to obtain maximum suppression of the H-reflex in D1 inhibition is approximately 20 ms, and many studies have used 20 or 21 msThe findings of the present study suggest that cycling intervention in adults with CP can induce suppression of H-reflex and stretch reflex in the leg, and activation of presynaptic inhibitory networks within the spinal cord. Therefore, leg cycling may reduce spasticity in the lower extremities of patients with CP.We are grateful to Dr. Megumi Toki, Assistant Professor of Sapporo Medical University, for her important contributions to the experiments. This study was supported by the Japanese Physical Therapy Association (JPTA H28-B39).The authors have no conflicts of interest in relation to this manuscript.


Regulation of amino acid’s biosynthetic pathway is of significant importance to maintain homeostasis and cell functions. Amino acids regulate their biosynthetic pathway by end-product feedback inhibition of enzymes catalyzing committed steps of a pathway. Discovery of new feedback resistant enzyme variants to enhance industrial production of amino acids is a key objective in industrial biotechnology. Deregulation of feedback inhibition has been achieved for various enzymes using in vitro and in silico mutagenesis techniques. As enzyme’s function, its substrate binding capacity, catalysis activity, regulation and stability are dependent on its structural characteristics, here, we provide detailed structural analysis of all feedback sensitive enzyme targets in amino acid biosynthetic pathways. Current review summarizes information regarding structural characteristics of various enzyme targets and effect of mutations on their structures and functions especially in terms of deregulation of feedback inhibition. Furthermore, applicability of various experimental as well as computational mutagenesis techniques to accomplish feedback resistance has also been discussed in detail to have an insight into various aspects of research work reported in this particular field of study. Product feedback inhibition of allosteric enzymes is of paramount importance in biotechnological industries for discovery of efficient microbial strains for increased production of metabolites of interest. Discovery of product-feedback inhibition traced back to 1950’s and was first reported by Novick and Szilard for the tryptophan biosynthetic pathway and has been reported as cornerstone for regulation of cell functions and control fluxes for optimal growth in microorganisms . AllosteExtensive studies have been reported for deregulation of feedback inhibition of various amino acids biosynthetic pathways using structural information of target enzyme, in vitro and in silico mutagenesis techniques. Most allosterically regulated enzymes are oligomeric in structural composition (i.e. made up of two or more polypeptide chains) having more than one active site and allosteric sites. Although reviews over the metabolic engineering strategies of amino acid biosynthetic pathways have been reported previously  still stRegulation of enzyme activity has profound effects on cell functions with huge practical applications. Enzyme activities can be regulated either by dissociation or binding of regulators and effectors causing conformational or structural changes that ultimately determine their catalytic activity , 9. SmalAllosteric regulation controls given protein activity involved in catalysis, signal transduction, gene regulation alongside various other biological processes , 15. RegInitially, the allosteric property was reported in quaternary proteins but later it has been confirmed as intrinsic characteristic of all dynamic proteins. Keeping in view the existence of these proteins as collection of active and inactive conformers, the binding of allosteric regulators causes structural changes in proteins and shifts this dynamic equilibrium either towards an active state  or inactive state  , 21 as dAmino acids are building blocks of proteins and are critical for life as they play role in synthesis of various metabolites through metabolic pathways. Amino acids biosynthesis is a complex array of alternate pathways connected as non-linear series of reactions . Apart fThe biosynthetic pathways for the essential amino acids  are found only in microorganisms and are more complex as compared to non-essential amino acids. Owing to their common metabolic precursors, the amino acids have been classified as four families namely Aspartate family  –30, PyruAvailability of given amino acids in living organisms are effected by either regulatory factors being capable of controlling synthesis of amino acids or either their proficient catabolism . So far,Different metabolic pathways involved in synthesis of amino acids are glycolysis; for branched chain amino acids  alongside Ala, Gly, Cys, Ser synthesis, citric acid cycle; for Asn, Asp, Lys, Met, Thr and Ile, phosphoribosyl pyrophosphate (PRPP) pathway; for aromatic amino acid  and histidine synthesis and shikimate pathway , 39. TheDespite the role of amino acids for accurate functioning of cells, homeostasis is maintained through metabolic regulation of biosynthetic pathways achieved either through (i) controlled synthesis of enzyme or (ii) feedback inhibition of enzyme by end product (i.e. amino acid). This end product inhibition of enzymes is counteracted either by sufficient availability of substrate or by heavy metal cation to facilitate balanced concentration of given metabolite/intermediate/amino acid for proper functioning of cell , 43, 44.Discovery of product feedback inhibition dated back to 1950, has been attributed as cornerstone of metabolic regulation that has been reported to sufficiently control metabolic fluxes to facilitate proficient growth . For a gE.coli through mutations at inhibitor binding site and identified E. coli MG1655 strain with improved L-lysine production yield by 46%. They utilized structural characteristics of L-lysine-sensitive DHDPS E.coli and L-lysine-insensitive DHDPSC.glutamicum and reported new enzyme variants through point mutations at specified sites with improved lysine production [S. marcescens .Few amino acids have the tendency to feedback inhibit multiple enzyme targets and their deregulation signifies their role to improve industrial production by identifying new and better strains. For instance, arginine and proline synthesized as final product from oxaloacetate pathway feedback inhibit N-acetyl-L-glutamate kinase (NAGK) and N-acetyl-L-glutamate synthase (in case of arginine) and glutamate kinase in case of proline . Similaroduction . Similaroduction . RecentlHere, we summarized structural characteristics of various enzyme targets and effect of mutations on their structures and functions especially in terms of deregulation of feedback inhibition. Applicability of various experimental as well as computational techniques  to accomplish feedback resistance has also been discussed in details to have an insight into various aspects of research work reported in this particular field of study.The amino acids can be broadly grouped as four categories or families following different pathways for their synthesis. The details of amino acids capable of inhibiting committed step of their own biosynthesis, their categories, pathways as per their common precursors and their feedback sensitive enzyme targets are shown schematically in Fig. Detailed structural features of various enzymes belonging to different families of amino acids have been provided in next section.E.coli follows linear route where hydrolysis of N-acetylornithine directly lead to production of ornithine and first enzyme N-acetylglutamate synthase (NAGS) is feedback inhibited by arginine and catalyzes rate determining step of route [P. aeruginosa follow a different route where acetyl group from acetylornithine is recycled to glutamate and in this case the N-acetylglutamate kinase (NAGK) is feedback inhibited by arginine [N-acetylglutamate kinase (NAGK) may serve as arginine-inhibitable or arginine-insensitive systems in different organisms.L-arginine has huge significance at industrial level especially, cosmetic industry, pharmaceutical and food industry. Microbial fermentation is employed for synthesis of arginine at industrial scale , 72, 73.of route . Few micarginine . Anotherarginine . The N-aE. coli and other microorganisms following linear route is catalyzed by N-acetyl-l-glutamate synthase (NAGS) that is inhibited by arginine [α helices [M. tuberculosis and N. gonorrhea provided insights into conformational arrangements needed for inhibition and regulation of enzyme activity and function. Both NAGSM. tuberculosis  connecting α4 and β6. The arrangement of amino acids involved at inter-junction of two monomer units to form dimer structure are depicted in Fig. First step of arginine biosynthetic pathway in arginine . Amino a helices , 79. ComDD, 5YO2  and NAGSDD, 5YO2  crystal N-acetyl-l-glutamate synthase (NAGS) belonging to N. gonorrhoeae i.e. l-arginine bound T-form (inactive) and R-form (active) with bound CoA and L-glutamate [Sequence alignment for NAGS sequences retrieved from different organisms revealed fully conserved and partially conserved amino acids around L-arginine-binding site suggesting role of key amino acids for binding of arginine. Mutations at these sites most probably deregulate feedback inhibition caused by arginine and will aid in discovery of feedback resistant strains with improved production of L-arginine. Li et al. reported comparison of two forms of lutamate , 81, 82.Saccharomyces cerevisiae, the NAGS and NAGK play their role as complex and are targeted by feedback inhibition of arginine [Maricaulis maris and Xanthomonas campestris have capability to oligomerize as tetramer where both NAT and AAK domains are similar to NAGS of N. gonorrhoeae but differences are observed for linker between two domains as well as their relative orientations [In case of Yeast like arginine , 83, 84.ntations . Furtherntations .l-arginine biosynthetic pathway as in case of Corynebacteria where acetyl group of N-acetyl-ornithine is recycled to generate l-glutamate [N-acetyl-l-glutamate kinase (NAGK) also termed as argB is rate limiting enzyme of cyclic route for lutamate , 73, 87.lutamate , 89. In lutamate .The key structural features of NAGK  from various microorganisms have been summarized in Tables E. coli are homodimer while those arginine sensitive type of NAGK are hexamer comprising of three homodimeric units structurally similar to NAGK of E. coli. Arginine feedback inhibition is not necessarily attributed to hexameric form but hexamer system play major role for proper functioning and stability of NAGK and enhances arginine sensitivity [Arginine insensitive NAGK belonging to sitivity .O-acetyltransferase (HTA) is feedback inhibited by methionine.Aspartate family includes lysine, threonine and methionine that regulate their own synthesis by end product feedback inhibition. Lysine and threonine synthesis is achieved both by DAP biosynthetic pathway or AAA biosynthetic pathway and enzymes catalyzing committed step of these routes are feedback inhibited by end product amino acids. The key enzyme targeted by lysine feedback inhibition are dihydrodipicolinate synthase (DHDPS), aspartokinase (AK) and while threonine feedback inhibit Homoserine kinase (HSK). Similarly, Homoserine Aspartokinase III The aspartate kinase the first enzyme of aspartate pathway is feedback inhibited by l-lysine in C. glutamicum. Three forms of aspartokinase  have been reported in different bacterial species. In case of B. subtilis and E. coli, the aspartokinase II and aspartokinase III are feedback inhibited by l-lysine and l-threonine [hreonine , 96.C. glutamicum. It is inhibited by l-lysine plus l-threonine; either l-lysine or l-threonine alone does not inhibit aspartokinase. In E. coli, many enzymes of lysine biosynthesis are regulated by the end-products of the pathway. There are three isozymes of aspartokinase in E. coli: aspartokinase I is repressed by l-threonine plus l-isoleucine and inhibited by l-threonine, aspartokinase II is repressed by l-methionine, and aspartokinase III is repressed and inhibited by l-lysine. Aspartokinase I and II are bifunctional enzymes and show both aspartokinase and homoserine dehydrogenase activities [The regulation of lysine biosynthesis varies among organisms. Only aspartokinase is regulated for the biosynthesis of lysine in tivities –99.C. glutamicum and E.coli [Comparing crystal structure (retrieved from protein data bank) of aspartokinase from different microorganisms namely d E.coli , 101 revC. glutamicum and AKE.coli identified presence of two state conformation (T-state and R-state) in case of E. coli where each monomer unit is comprised of two ACT domain at C-terminus while each monomer unit of tetramer structure of C. glutamicum has single ACT domain [C. glutamicum is feedback inhibited by both lysine and threonine in a concerted way while AK of T. thermophilus is feedback inhibited by threonine only [C. glutamicum as: inhibitory complex bound to lysine and threonine, (ii). Active complex bound to threonine. Comparison of these two forms of AK revealed that inhibitory form is stabilized by conformational changes caused by binding of inhibitors. Structure of AK is heterotetramer comprised of two α subunits and two β subunits where dimerization of homo-oligomeric AK involves interaction among regulatory domains. The crystal structure with bound lysine and threonine exists as in active T-form [Comparison of AKT domain , 102. Thine only –105. Ayae T-form . AK struHistidine biosynthetic pathway found in bacteria, plants and fungi comprises of ten steps and has been considered as well recognized target for discovery of various antimicrobial drugs. The enzyme ATP-phosphoribosyl transferase (ATP-PRT) catalyzing first committed step of pathway is feedback inhibited by end product histidine. The reaction catalyzed by ATP-PRT involves synthesis of N′-5′-phosphoribosyl-ATP (PR-ATP) by condensation of ATP with 5-phosphoribosyl-a-1-pyrophosphate (PRPP) . The enzM. tuberculosis is homo-hexameric class, also found in S. enterica and E. coli [β-strands arranged as three domains. Domain I contains a central β-sheet consisting of four parallel β-strands and two anti-parallel strands while domain II also has four parallel β-strands and one anti-parallel β-strand surrounded by two α-helices on each side. Catalytic site (substrate binding site) of ATP-PRT lies at intersection of domain I and II involving mostly interaction with amino acids of domain II while allosteric inhibitor histidine binds in domain III. The catalytic domain of ATP-PRT of both mycobacteria and glutamine binding E. coli are similar to each other [Crystal structural analysis of tertiary and quaternary ATP-PRT revealed two different forms. The most widely found homo-hexameric form, encountered in bacteria, fungi, and plants, while second form i.e. the hetero-octameric type, limited to few bacterial strains. ATP-PRT belonging to  E. coli , 108. Cr E. coli . Further E. coli . The mycch other , 109.The ATP-PRT enzyme of proteobacteria has shorter form without domain III where HisG forms complex with HisZ. Crystal structure of both apo and AMP:His complex is in hexamer form comprised of trimer of dimers where histidine bound hexameric complex is more compact than apo form as shown in Fig. Histidine binding alongside AMP causes conformational changes in ATP-PRT structure where active site of enzyme is closed and amino acid residues involved in binding of PRPP also disrupt that hindered catalytic activity .l-phenylalanine and l-tyrosine are formed from chorismic acid via prephenate, which undergoes either decarboxylation/dehydration or decarboxylation/dehydrogenation, followed by a transamination to generate given amino acids. The enzymes namely prephenate dehydrogenase and prephenate dehydratase catalyzes first step of tyrosine pathway and phenylalanine pathway, respectively and are feedback inhibited by their end product.Aromatic amino acids namely tryptophan, phenylalanine and tyrosine follow Shikimate pathway for their biosynthesis. Despite their role as building block for protein, they also serve as precursor for various pharmaceutical products. For instance, Tryptophan serve as precursor for various metabolites with significant pharmacological importance especially, vinblastine and vincristine two well-known anticancer drugs while Tyrosine is precursor for neurotransmitter dopamine, p-hydroxystyrene and p-hydroxycinnamic acid, needed for manufacturing of novel materials, pharmaceuticals and nutraceuticals –115. Cho2:TrpG2 and catalyzes the first reaction branching from the shikimate pathway toward the biosynthesis of tryptophan occurring in bacteria, fungi and plants [3 while TrpG belongs to glutamine amidotransferase family [α-subunit or both subunits [Serratia marcescens in the presence of its substrates and inhibitor l-tryptophan. The trpE of S. marcescens comprises of domain I  and domain II  alongside two α-helices . The junction of two domains contains various amino acids causing tryptophan feedback inhibition while another neighboring crevice attached to trpG contains amino acid residues involved in substrate binding [Tryptophan belong to aromatic amino acids family and its biosynthesis proceeds via a common pathway to chorismate . The fird plants . AS is ce family . The reasubunits . Glen et binding .S. marcesens is comparable to AS from S. solfataricus while AS of S. typhimurium represents different structure as depicted in Fig. Comparison of crystal structures of AS from various organisms revealed different arrangement of TrpG and TrpE in heterotetrameric complex. The structural composition of AS from S. marcescens is comparable to S. solfataricus where Gly328, Thr329, His398 and Gly485 from chorismate binding domain play vital role in binding of chorismate while Ser40, Pro291, Met293, Val453, Tyr455 are involved in binding of tryptophan. Similarly, glutamine, pyruvate and benzoate binding site are also shown as different regions in Fig. α- and β) of anthranilate synthase involving reaction of chorismate with glutamine while only α-subunit is needed for reaction of chorismate and ammonia (at high concentrations of ammonium) [Structural characteristics of TrpG and TrpE from mmonium) , 122.α-helices and C-terminal domain formed by eight α-helices surrounding seven stranded β-sheet [S. solfataricus, P. carotovorum, T. thermophiles, X. campestris, M. tuberculosis [The second enzyme of tryptophan biosynthetic pathway anthranilate phosphoribosyl transferase (AnPRT) is also feedback inhibited by L-tryptophan. The AnPRT (TrpD) catalyzes reaction of PRPP and anthranilate to N-(5′-phosphoribosyl)-anthranilate (PRA) and PPi. Crystal structure revealed AnPRT as homodimer having N-terminal domain comprising of six  β-sheet . The actrculosis –128.The enzyme anthranilate phosphoribosyl transferase (AnPRT) belonging to enteric bacteria exists as complex with anthranilate synthase (AS). Bifunctional TrpD: TrpE complex is responsible for the first step in tryptophan biosynthesis i.e. the transfer of an amino group from glutamine to chorismate and the formation of anthranilate. The Feedback inhibition of anthranilate phosphoribosyl transferase (AnPRT) is observed in its complex form with AS and sensitivity to tryptophan feedback inhibition is lost once complex is distorted .α-isopropylmalate catalyzed by α-isopropylmalate synthase (α-IPMS) regulated by leucine feedback inhibition [The branched chain amino acids follow pyruvate pathway for their synthesis and use pyruvate or 2-ketobutyrate as precursor formed through acetohydroxy acid synthase (AHAS). The pyruvate pathway branches at isopropylmalate (IPM) where 2-ketoisovalerate and acetyl CoA is converted to hibition . The IPMhibition –133.α-isopropylmalate synthase catalyzing first committed step of leucine biosynthetic pathway branched from pyruvate route is feedback inhibited by end product leucine. The reaction catalyzed by IPMS involves Claisen condensation of α-ketoisovalerate and AcCoA to produce α-isopropylmalate [α-IPMS from M. tuberculosis, a monomeric structure comprising of two domains i.e. N-terminal  and C-terminal (regulatory site) connected through two subdomains depicted in Fig. α/β)8 TIM barrel where linker domain I comprises of α10 and two short β-strands, while subdomain II composed of α11-α13. Similarly, regulatory domain located at C-terminal is comprised of two βββα units. They also identified that C-terminal domain markedly effect activity of enzyme [The ylmalate . Luiz etf enzyme , 136.Figα-IPMS revealed binding of Zn2+ and α-ketoisovalerate into active site while leucine exhibits reversible feedback inhibition by binding inside regulatory domain. Feedback inhibition of α-IPMS by leucine follow time dependent slow onset inhibition where complex formed by leucine binding inside regulatory domain isomerizes to tightly bound complex. The linker domain facilitate in transmission of inhibitory signals towards catalytic site [α-IPMS from S. cerevisiae mutations at leucine binding site [α-IPMSMtbTyr410Phe mutant also showed insensitivity to leucine binding [Structural analysis of tic site . Cavalieing site  while an binding .It is a generalized fact that structural characteristics of given enzyme system has marked effect on deregulation of feedback inhibition of amino acids. Enzymes existing as monomer, dimer or oligomer complexes comprised of different domains, chains, and sites. Mutations at any of these sites negatively effects binding of inhibitors and ultimately inhibition tendency alongside catalytic activity of that enzyme complex is disrupted. Here, we discussed applicability of various mechanisms and strategies employed to accomplish deregulation of feedback inhibition for histidine biosynthetic pathway (i.e. taken as example).Histidine has huge significance as per its role in various pharmaceutical products and also serve as precursor for production of various bioactive compounds. Owing to its role in various industries like pharmaceutical and cosmetic industry, the aim of biotechnologists is to enhance its production at industrial scale by implementing various available techniques and approaches. Bioinformatician, as well as experimentalists are contributing their efforts to accomplish deregulation of enzyme′s feedback inhibition. Commonly used strategies are (1). Reducing interaction and binding of feedback inhibitor inside pocket, (2). Blocking entry of inhibitor into binding domain, (3). Mutations at other parts/sites of enzyme to eliminate metastability of protein due to inhibitor binding, (4). Effect of mutations at interface of dimer or multimer enzyme, (5). Competitive feedback inhibition by end product (i.e. amino acid). The structural characteristics of ATP-phosphoribosyl transferase (ATP-PRT) enzyme (feedback inhibited by end product histidine) revealed two forms i.e. homo-hexamer or hetero-octamer. Detailed discussion over use of various techniques and strategies for deregulation of feedback inhibition in HisG are provided in next section.Reducing binding of feedback inhibitor inside pocket/regulatory domain Allosteric inhibitor binding sites have been previously engineered by various research groups to accomplish reduced binding of inhibitor inside pocket. Reducing interaction tendency of inhibitor by mutations of key amino acids of binding domain is of paramount importance for deregulation of feedback inhibition. Cho et al., reported crystal structure of ATP-Phosphoribosyl transferase (HisG) and identified key amino acids involved in binding of allosteric inhibitor histidine [M. tuberculosis and C. glutamicum. The N215K/L231F/T235A mutant of HisG resulted in higher yield of histidine [C. glutamicum where four amino acids of regulatory domain directly interacting with l-lysine were mutated by Feng et al. [l-lysine binding and inhibition. As mentioned earlier, the histidine binding into allosteric site (Domain III) of ATP-PRT results in more compact and inactive form of enzyme. Identifying key amino acids involved in binding of L-histidine like Asp218, Thr238, and Ala273 [E. coli AKIII (lysine-insensitive AKIII) were reported by Falco et al. [istidine . Zhang eistidine . Similaristidine . Similarg et al. . They red Ala273  and sequd Ala273  while Meo et al. .C. crenatum by mutations at Glu19 position located at entrance of arginine binding site. Where the feedback inhibition of l-arginine was most deregulated in the Glu19Tyr, Glu19Trp and Glu19Phe mutants [Blocking entry of inhibitor into binding domain Modifying the key amino acid residues positioned at the entrance of feedback inhibitor binding site facilitate to block entry of inhibitor Fig.  mutants . Now, inEliminate metastability of protein due to inhibitor binding As histidine binding introduces compactness in trimeric, or multimeric structure of ATP-PRT enzyme. Mutations or engineering of sites other than feedback inhibitor binding site may cause conformational changes alongside disruption of enzyme stability that ultimately destroy catalytic activity of enzyme. Owing to effect of histidine binding with key amino acids of binding domain, the modifications on other sites or positions of enzyme cause conformational changes and allosterically effect catalytic activity as well as inhibitor binding tendency of ATP-PRT. Plenty of approaches i.e. both experimental and computational are in common practice to introduce mutations in enzyme structure for discovery of new enzyme variants. This particular approach has been successfully employed by various research groups for aspartokinase III to identify enzyme mutant structures with deregulated inhibition for instance Gln298Gly and Ala279Thr (using point mutation approach) [pproach) –176.Effect of mutations at interface of dimer or multimer enzyme Histidine need ATP-PRT enzyme in trimer or hexamer form to show its feedback inhibition as Histidine binding domain lie at junction of two monomeric units. Mutations at interface of dimer structure may disrupt inter-domain communication and transfer of signals leading to loss of catalytic activity and feedback inhibition as shown in Fig. α-IPMS (mutant Y410F) with complete loss of sensitivity towards feedback inhibition by leucine [ leucine .As histidine has its own binding domain/regulatory domain and its interactions with pocket affects catalytic activity through allosteric effect. On the other hand, few amino acids like L-threonine showed feedback inhibition as competitive inhibitor and disrupting their binding inside active pocket will ultimately destroy the catalytic activity. Multiple strategies could be employed to retain wild-type activity while decreasing affinity for the competitive inhibitor. Like engineer new interactions that support substrate binding but weaken inhibitor binding, effecting hydrophobicity, creating steric bulk or decreasing strength of van dar Waals interactions.Plenty of mutagenesis approaches including both experimental and computational techniques are in common practice to achieve deregulation in targets of interest. Commonly used in vitro mutagenesis techniques are Directed evolution technique , 178, SiNo significant studies have been reported for mutational studies over Anthranilate synthase (AS), Anthranilate phophoribosyl transferase (AS-PRT), Pyrroline carboxylase reductase, Glutamate 5-kinase (GK), and Homoserine-O-acetyltransferase (HSAT) enzyme targets regulated by feedback inhibition of their own end product amino acids.In addition, the development of bioinformatics algorithms enabled computational approaches to provide more precise guidance for enzyme engineering and make it more efficient and less laborious. The success of rational design depends on in-depth knowledge about sequence and structure features of target proteins. Various computational mutagenesis approaches have been reported to get in depth sights of protein functions, structure, stability and thermodynamic characteristics Table . The folLiterature revealed that none of above mentioned in silico mutagenesis techniques have been employed for mutations prediction to deregulate feedback inhibition of enzymes. Using combination of various in silico approaches to design new enzyme variants with deregulated feedback inhibition is of worth importance.Current review emphasized need of new mutagenesis techniques to accomplish more efficient enzyme variants aimed for increased production of amino acids. The detailed description of enzyme structures here affirmed that (a). Engineering of regulatory domain is key contributor to accomplish deregulation of feedback inhibition, (b). Blocking entry of inhibitor into binding domain by modifying the key amino acid residues positioned at the entrance of feedback inhibitor binding site, steric hindrance and charge modification is of paramount importance for deregulation, (c). In case of dimer, tetramer or hexameric structures, mutations at interface or junction of two monomeric units may disrupt signal transfer and ultimately destroy enzyme activity.Biotechnologists, microbiologists and other research groups need to develop new strategies and approaches with better efficacy. Rational mutagenesis technique like site directed mutagenesis (SDM) and site saturation mutagenesis (SSM) have many advantages over random mutagenesis approach. Amalgamation of in vitro and in silico would be more effective approach and focus should be on more reliable and efficient software designs.Detailed literature search over amino acid biosynthetic pathways, structural details of target enzymes feedback inhibited by amino acid, mutagenesis approaches (both in vitro and in silico) used to incorporate structural and conformational changes to deregulate their inhibition tendency have been summarized. The structure, design and mechanism of product feedback inhibition for all enzyme targets have been discussed in detail to provide an insight into structural and functional basis of amino acid feedback inhibition. Bacterial amino acids biosynthetic pathways are interconnected as different amino acids may be produced using same common precursor as starting point thus has been categorized into various groups. Majority of amino acid’s biosynthetic pathways (16 out of 20 amino acids) are regulated by allosteric feedback inhibition. Structural analysis of enzyme targets that are feedback inhibited by their own end product revealed that they exists mostly as dimer, tetramer or hexamer structure comprising of multiple subunits having more than one binding and allosteric sites. Although some enzyme targets also exist as monomer structures but allosteric inhibition is mostly observed in dimer or oligomeric systems. Traditional methods used for discovery of new bacterial strains like random mutagenesis have been replaced by rational mutagenesis approach and plenty of new computer assisted methods are in use now. Identifying key sites and domains in given enzyme structure keeping in view their role in feedback inhibition prior to mutations is of worth importance that will facilitate new enzyme discovery efficiently. Current review will provide guidelines for designing of better feedback resistant enzymes and will facilitate biotechnologists for discovery of novel enzyme variants for increased production of amino acids at industrial scale.


To clarify the wind veer characteristics with height and their effect on the wind turbine power outputs, an investigation was carried out at the wind farms with complex and simple terrains. A 2 MW and a 1.5 MW wind turbine were tested, each having an 80 m tall met mast and a ground lidar to capture wind veering. Wind veer conditions were divided into four types based on wind direction changes with height. The power deviation coefficient (PDC) and the revenue differences for the four types were derived from the estimated electric productions. As a result, the wind veer angle across turbine rotors were more significant at the complex site than at the simple site. For the two sites, the PDC values ranged from − 3.90 to 4.21% depending on the four types, which led to a 20-year revenue variation of − 274,750–423,670 USD/MW. Every site has unique conditions, thus wind measurement campaigns should be carried out to estimate accurate electric power productions of wind turbines at a given site. Over the last decades, the investigations on the effect of atmospheric stability conditions on wind power productions have been conducted by many researchers6. Recently, the wind directional change with height has been given much attention by wind energy industries since it influences turbine productions, however, this relation is not widely studied compared to other atmospheric parameters.The power outputs of a wind turbine have been known to be affected by the factors of incoming wind speed, wind direction, atmospheric stability, turbulence, air density, wind shear, wind veer and so on. The factors influencing wind turbine power production have a diurnal cycle due to the surface heat from the sun8. The veering wind generally occurs in the stable atmospheric condition, while the backing wind occurs in the unstable atmospheric condition10. Terrain characteristics also have an influence on directional wind shear. The wind veer angles of the hilly terrain condition were much higher than those of the open sea condition11. Also, the wind veer angles on the lower part of the rotor tend to have higher values than those on the upper part on sites with complex terrain12.When the wind direction changes with height above the ground, it is referred to as wind directional shear or wind veer. It is a veering wind when the wind direction changes clockwise with height and it is a backing wind when the wind direction changes counter clockwise with height14 as well as mechanical load16. The effect of the overall wind veer not divided by veering and backing winds has been analyzed by Saint-Drenan et al.17 and Bardal et al.18, whose results showed that the effect of wind veer on turbine power performance was comparably small. The impact of power production has been studied with the feature of veering and backing winds20. It was pointed out that a small clockwise directional shear caused an increase of the power output while a counter clockwise directional shear resulted in underperformance. Murphy et al.21 also obtained that a veering wind led to power gain while a backing wind resulted in power loss.A large wind turbine could experience a comparatively large wind directional difference across the rotor. Several studies have been carried out to determine the influence of the directional shear on wind turbine power performance. It was reported that wind directional differences had an effect on wind turbine power output22. It was found that the power deviation coefficient was in a range from − 6.5 to 1.6% for the four types. Further investigation is necessary on wind veer characteristics with various wind farm sites.Although most of the studies on wind veer characteristics were carried out in terms of veering and backing winds, various wind veer conditions across the rotor should be considered since topographical and roughness conditions will vary at many sites. Recently, the power deviation coefficient was computed in accordance with the four types with veering conditions at upper and lower areas from the turbine hub heightThis work aims to investigate the relationship between wind veer characteristics and the power output at two wind farm sites: one near a large landfill and the other located at the seaside. The wind veer was classified by four types depending on the wind direction change across the rotor with height above the ground. The power curves from the four types were compared with that under no veer condition to reveal the power deviation. The revenue differences along with various electricity productions with the four types were analyzed according to annual average wind speeds from 4 to 11 m/s.23:This work was carried out at wind farms on Jeju Island which is located off the southern part of the Korean peninsula. Figure 24 value of 0.87% was found in the measurement sector due to the nearby landfill at the DB site. On the other hand, the measurement sector of the HW site had an RIX value of 0.00% due to the very simple terrain and the seaside.The measurement sectors were from 273° to 93° at the DB site and 301° to 47° at the HW site, respectively. The comparatively complex terrain with the ruggedness index (RIX)Table 25 module for ground lidar was used at the DB site to correct the wind measurement error caused by the terrain complexity. However, it was not used at the HW site as the measurement site was considered to have a simple terrain. The power outputs from the test wind turbines were collected through the Gateway Supervisory Control and Data Acquisition (SCADA) system.The air temperature, the humidity and the atmospheric pressure data were used for normalization of the specific air density at the two sites. The data were measured at a height of 75 m at both sites using the meteorological sensors listed in Table 26 and the ground lidar user manual27. Also, when the wind turbines produce electric power significantly lower or higher than the expected power, or when the wind turbines were in abnormal operation, or when the turbine yaw misalignment was over 30°28, the wind measurements and the power data were removed from the database. Additionally, not only the wind speeds lower than the cut-in and higher than the cut-out speeds but also the data outside the measurement sector were discarded for the analysis.The wind data with carrier-to-noise ratios (CNR) of less than − 23 db or with an availability of less than 80% or data measured by lidar in abnormal operation were rejected according to the study by Kim et al.After the filtering process, the remaining data were approximately 50.7% for the DB site and 26.7% for the HW site, respectively, which was named as all data for formal analysis in this work.b) by the following equation30:1 and T2 are the air temperatures at the 75 m and 3 m heights at both sites in this work, respectively. The atmosphere is under an unstable condition when the value of Rib is lower than − 0.02. When the value is higher than 0.02, the atmosphere is under a stable condition. Also, the values between − 0.02 and 0.02 correspond to a neutral atmospheric condition. The atmospheric stability conditions at the DB site were categorized into comparatively fair ratios with 34.9%, 41.5% and 23.5% for stable, neutral and unstable conditions, respectively. On the other hand, the HW site had prevalent neutral conditions with 68.1%, followed by stable and unstable conditions with 20.5% and 11.4%, respectively. The two sites had typical features of the onshore and offshore atmospheric stability conditions, which is a similar portion for the land and prevailing condition of neutral for the sea32.The fundamental analysis on wind conditions was performed for the two sites. The atmospheric stability conditions were evaluated in terms of the bulk Richardson number  of 0.996 and 0.977 at the DB and the HW sites, respectively. Although more scattering was observed at the HW site, the difference between the values of R2 for the two sites were very low. This meant that very small measurement error occurred at the HW site so that the measurements at both sites were used without any data correction in this work.The results of the linear regression analysis between the met mast and the ground lidar wind speeds at the two sites are displayed in Fig. Figure 35. The wind veer is evaluated by the following equation36:According to the characteristics of the wind direction change with height above the ground, the wind veering phenomenon can be categorized as a veering or backing wind. When the wind direction shifts clockwise with an increase of height, the wind is referred to as a veering wind. On the other hand, it is a backing wind when the wind direction changes counter clockwise with an increase of height12 and Rybchuk et al.37.The wind veer was analyzed using the wind directions at the measurement heights of 40 m, 80 m and 124 m at the DB site and 40 m, 70 m and 115 m at the HW site, respectively. Table 22. The four types of wind veer conditions are illustrated in Fig. The wind veer types were divided by the wind direction changes from the upper part of the swept area to the hub height and from the hub height to the lower part of the swept areaFigure Figure As for the diurnal frequency, at the DB site, the types VV and BV exhibited opposite diurnal variations, while the other two types had similar diurnal frequencies throughout the day. In contrast, at the HW site, there were small variations observed for all the types for diurnal frequency regardless of daytime or nighttime. This result was considered to be caused by the considerable diurnal variation of atmospheric stability at the DB site and little variation at the HW site, as shown in Fig. Figure The wind veer values and fluctuations of the types VV and BB at the DB site were higher during the nighttime than during the daytime. On the other hand, little change of wind veer values and fluctuation were found for the remaining two types, VB and BV, throughout the day at the HW site, since the two types contained both positive and negative wind veer angles, as shown in Fig. 22.The diurnal variation of wind veer on the upper and lower parts of the rotor are compared in Fig. 12.The comparatively complex terrain at the DB site had more effect on wind veer on the upper and lower parts than the very simple terrain and the seaside at the HW site. The wind veer values on the lower part at the DB site were more influenced by the topographical condition compared to those on the upper part. From the error bar at both sites, the wind veer fluctuation of the lower part was more significant than the upper part. Therefore, the wind veer shifting angle with height could rely on the topographical condition. In other words, a complex terrain could have more significant wind veer angle than a simple terrain and furthermore the shifting angle could be bigger at the lower part than at the upper part of a turbine rotor. This finding was similar to the work of Wharton et al.21. For this reason, the types VV and BB also had higher values and fluctuations at wind speeds less than 6 m/s. On the other hand, the types VB and BV showed small wind veer values as well as little fluctuations at all wind speeds due to the neutralization effect.The wind veer variation with wind speeds for the four cases are presented in Fig. 21 and Shu et al.11.The wind veer variation with wind speeds for the upper and the lower part of the rotor are represented in Fig. 38. The power curve under no wind veer condition was derived using the data points that had wind veer values within ± 0.01 deg/m22. The wind data points with no veer condition accounted for 11.3% and 42.5% of all data points at the DB and the HW sites, respectively. The power curve under no veer condition was used as a reference for further analysis in this work.The measured wind speeds were normalized to standard air density and the power curves of the test wind turbines were drawn in compliance with IEC 61400-12-1 3rd editionFigure 22:each type is power outputs of each type and Pno veer is those under no veer condition.The power deviation coefficient (PDC) was calculated by the following equationThe PDC was computed for power outputs before the rated wind speed to minimize the neutralization by almost the same power outputs after the rated wind speed. The value of the PDC indicates power gain or power loss depending on the outputs higher or lower than the zero.19 and Murphy et al.21. On the other hand, Gao et al.22 and Gomez et al.20 reported that the veering wind led to power loss while power gain was found with the backing wind. Accordingly, the finding about power gain and loss in this work was very site specific and further research is needed.Figure each type and AEPno veer are annual energy production (AEP) of each type and that under no veer condition, respectively. REC is the renewable energy certificate price, SMP is the system marginal price, and Y is operating years. Prated is the rated power of the test wind turbines for normalization.The revenue difference by electric productions from the four types was evaluated at various annual average wind speeds. The revenue difference was calculated by the following equation:38. The fixed price for summation of REC and SMP was 135.9 US dollars (USD) per MWh as of January 2023 in South Korea, which was applied to this work.The AEP was calculated using the combination of a Rayleigh distribution for annual average wind speeds from 4 to 11 m/s and measured power curves according to the IEC 61400-12-1 3rd editionFigure The highest revenue difference was found at type BV at the HW site, which resulted from the corresponding higher power output difference, as shown in Fig. An 83.1% of dominant veering wind was observed at the DB site. The HW site had similar proportions of veering and backing winds with 53.1% and 46.9%, respectively. The type VV had the highest frequency of 62.7%, while the type VB was the smallest with 4.9% at the DB site. The frequencies of four types, VV, VB, BB and BV were 32.4%, 20.8%, 31.1% and 15.7% at the HW site, respectively.The topographical condition had more influence on wind veer characteristics at the lower part than the upper part at the DB site with comparatively complex terrain, while those effects were small at the HW site with simple terrain and the seaside.In this work, for both sites, the types VV and BV had positive PDC values ranging from 1.45 to 4.21% resulting in a power gain. On the other hand, the types VB and BB resulted in negative PDC values ranging from − 0.62 to − 3.90%, which led to power loss. More variation in PDC caused by more significant wind veering was found at the DB site with comparatively complex terrain than at the HW site with simple terrain.The highest revenue differences at both sites occurred at 6 m/s and 7 m/s, in which the types VV and BV had a positive revenue difference resulting from positive PDC, while the types BB and VB had a negative revenue difference coming from negative PDC. The 20-year revenue differences at the two sites were in the range of − 274,750 to 423,670 USD/MW relying on the four types of wind veer at the annual average wind speeds of 4 to 11 m/s.The results of this study can be summarized as follows: